Attenuated human-bovine chimeric parainfluenza virus (PIV) vaccines

ABSTRACT

Chimeric human-bovine parainfluenza viruses (PIVs) are infectious and attenuated in humans and other mammals and useful individually or in combination in vaccine formulations for eliciting an immune response to PIV or other pathogens. Also provided are isolated polynucleotide molecules and vectors incorporating a chimeric PIV genome or antigenome which includes a partial or complete human or bovine PIV “background” genome or antigenome combined or integrated with one or more heterologous gene(s) or genome segment(s) of a different PIV. Chimeric human-bovine PIV of the invention include a partial or complete “background” PIV genome or antigenome derived from or patterned after a human or bovine PIV virus combined with one or more heterologous gene(s) or genome segment(s) of a different pathogen, including different PIV virus to form the human-bovine chimeric PIV genome or antigenome.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority under 35 USC § 120 as aContinuation Application of U.S. application Ser. No. 11/078,658, filedMar. 11, 2005, now abandoned, which in turn is a ContinuationApplication of U.S. application Ser. No. 10/915,634, filed Aug. 10,2004, now abandoned, which in turn is a Continuation Application of U.S.application Ser. No. 10/030,544, filed Jun. 27, 2002, now abandoned,which in turn is a National Stage Application under 35 USC § 371 ofInternational Application PCT/US00/17066, filed Jun. 15, 2000, which inturn claims priority under 35 USC § 119(e) of U.S. ProvisionalApplication 60/143,134, filed Jul. 9, 1999. The present application alsoclaims priority under 35 USC § 120 as a Continuation-In-Part Applicationof U.S. application Ser. No. 09/458,813, filed Dec. 10, 1999, issued asU.S. Pat. No. 7,314,631 on Jan. 1, 2008, which in turn is aContinuation-In-Part Application of U.S. application Ser. No.09/083,793, filed May 22, 1998, issued as U.S. Pat. No. 7,208,161 onApr. 24, 2007, which in turn claims priority under 35 USC § 119(e) ofU.S. Provisional Application 60/059,385, filed Sep. 19, 1997, and U.S.Provisional Application 60/047,575, filed May 23, 1997. The presentapplication also claims priority under 35 USC § 120 as aContinuation-In-Part Application of U.S. application Ser. No.09/459,062, filed Dec. 10, 1999, issued as U.S. Pat. No. 7,250,171 onJul. 31, 2007, which in turn is a Continuation-In-Part Application ofU.S. application Ser. No. 09/083,793, filed May 22, 1998 and issued asU.S. Pat. No. 7,208,161 on Apr. 24, 2007, which in turn claims priorityunder 35 USC § 119(e) of U.S. Provisional Application 60/059,385, filedSep. 19, 1997 and U.S. Provisional Application 60/047,575, filed May 23,1997. The present application also claims priority under 35 USC § 120 asa Continuation-In-Part Application of U.S. application Ser. No.09/586,479, filed Jun. 1, 2000, and issued as U.S. Pat. No. 7,201,907 onApr. 10, 2007, which in turn is a Continuation-In-Part Application ofU.S. application Ser No. 09/083,793, filed May 22, 1998 and issued asU.S. Pat. No. 7,208,161 on Apr. 24, 2007, which in turn claims priorityunder 35 USC § 119(e) of U.S. Provisional Application 60/059,385, filedSep. 19, 1997and U.S. Provisional Application 60/047,575, filed May 23,1997.

BACKGROUND OF THE INVENTION

Human parainfluenza virus type 3 (HPIV3) is a common cause of seriouslower respiratory tract infection in infants and children less than oneyear of age. It Is second only to respiratory syncytial virus (RSV) as aleading cause of hospitalization for viral lower respiratory tractdisease in this age group (Collins et al., in B. N. Fields Virology, p.1205-1243, 3rd ed., vol. 1., Knipe et al., eds., Lippincott-RavenPublishers, Philadelphia, 1996; Crowe et al., Vaccine 13:415-421, 1995;Marx et al., J. Infect. Dis. 176:1423-1427, 1997, all incorporatedherein by reference). Infections by this virus result in substantialmorbidity in children less than 3 years of age. HPIV1 and HPIV2 are theprincipal etiologic agents of laryngotracheobronchitis (croup) and alsocan cause severe pneumonia and bronchiolitis (Collins et al., 1996,supra). In a long term study over a 20-year period, HPIV1, HPIV2, andHPIV3 were identified as etiologic agents for 6.0, 3.2, and 11.5%,respectively, of hospitalizations for respiratory tract diseaseaccounting in total for 18% of the hospitalizations, and, for thisreason, there is a need for an effective vaccine (Murphy et al., VirusRes. 11:1-15, 1988). The parainfluenza viruses have also been identifiedin a significant proportion of cases of virally-induced middle eareffusions in children with otitis media (Heikkinen et al., N. Engl. J.Med. 340:260-264, 1999, incorporated herein by reference). Thus, thereis a need to produce a vaccine against these viruses that can preventthe serious lower respiratory tract disease and the otitis media thataccompanies these HPIV infections. HPIV1, HPIV2, and HPIV3 are distinctserotypes that do not elicit significant cross-protective immunity. Themajor protective antigens of PIVs are the hemeagglutinin (HN) and fusion(F) glycoproteins, which mediate viral attachment, penetration andrelease. Protection against reinfection is mediated primarily byvirus-neutralizing antibodies.

Despite considerable efforts to develop effective vaccine therapiesagainst HPIV, approved vaccine agents that prevent HPIV related illnesshave not yet been achieved. The most promising prospects to date arelive attenuated vaccine viruses since these have been shown to beefficacious in non-human primates even in the presence of passivelytransferred antibodies, an experimental situation that simulates thatpresent in the very young infant who possesses maternally acquiredantibodies (Crowe et al., 1995, supra; and Durbin et al., J. Infect.Dis. 179:1345-1351, 1999a; each incorporated herein by reference). Twolive attenuated PIV3 vaccine candidates, a temperature-sensitive (ts)derivative of the wild type PIV3 JS strain (designated PIV3cp45) and abovine PIV3 (BPIV3) strain, are undergoing clinical evaluation (Karronet al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Karron et al., 1995a,supra; Karron et al., 1995b, supra; each incorporated herein byreference). The BPIV3 vaccine candidate is attenuated, geneticallystable and immunogenic in human infants and children. A second PIV3vaccine candidate, JS cp45, is a cold-adapted mutant of the JS wildtype(wt) strain of HPIV3 (Karron et al., 1995b, supra; and Belshe et al., J.Med. Virol. 10:235-242, 1982a; each incorporated herein by reference).This live, attenuated, cold-passaged (cp) PIV3 vaccine candidateexhibits temperature-sensitive (ts), cold-adaptation (ca), andattenuation (att) phenotypes, which are stable after viral replicationin vivo. The cp45 virus is protective against human PIV3 challenge inexperimental animals and is attenuated, genetically stable, andimmunogenic in seronegative human infants and children (Belshe et al.,1982a, supra; Belshe et al., Infect. Immun. 37:160-165, 1982b; Clementset al., J. Clin. Microbiol. 29:1175-1182, 1991; Crookshanks et al., J.Med. Virol. 13:243-249, 1984; Hall et al., Virus Res. 22:173-184, 1992;Karron et al., 1995b, supra; each incorporated herein by reference).Because these PIV3 candidate vaccine viruses are biologically derivedthere is no proven method for adjusting their level of attenuation aswill likely be necessary for broad clinical application.

To facilitate development of PIV vaccine candidates, recombinant DNAtechnology has recently made it possible to recover infectiousnegative-strand RNA viruses from cDNA (for reviews, see Conzelmann, J.Gen. Virol. 77:381-389, 1996; Palese et al., Proc. Natl. Acad. Sci.U.S.A. 93:11354-11358, 1996; each incorporated herein by reference). Inthis context, rescue of recombinant viruses has been reported forinfectious respiratory syncytial virus (RSV), rabies virus (RaV), simianvirus 5 (SV5), rinderpest virus, Newcastle disease virus (NDV),vesicular stomatitis virus (VSV), measles virus (MeV), mumps virus (MuV)and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presenceof essential viral proteins (see, e.g., Garcin et al., EMBO J.14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A.92:4477-4481, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnellet al., EMBO J. 13:4195-4203, 1994; Whelan et al., Proc. Natl. Acad.Sci. U.S.A. 92:8388-8392, 1995; Hoffman et al., J. Virol. 71:4272-4277,1997; Kato et al., Genes to Cells 1:569-579, 1996, Roberts et al.,Virology 247:1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997;International Publication No. WO 97/06270; Collins et al., Proc. Natl.Acad. Sci. USA 92:11563-11567, 1995; U.S. patent application Ser. No.08/892,403, filed Jul. 15, 1997 (corresponding to publishedInternational Application No. WO 98/02530 and priority U.S. ProvisionalApplication Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May9, 1997, and 60/021,773, filed July 15, 1996); U.S. patent applicationSer. No. 09/291,894, filed on Apr. 13, 1999; U.S. Provisional PatentApplication Ser. No. 60/129,006, filed Apr. 13, 1999; U.S. ProvisionalPatent Application Ser. No. 60/143,097, filed by Bucholz et al. on Jul.9, 1999; Juhasz et al., J. Virol. 71:5814-5819, 1997; He et al. Virology237:249-260, 1997; Peters et al. J. Virol. 73:5001-5009, 1999; Whiteheadet al., Virology 247:232-239, 1998a; Whitehead et al., J. Virol.72:4467-4471, 1998b; Jin et al. Virology 251.:206-214, 1998; Bucholz etal. J. Virol. 73:251-259, 1999; Whitehead et al., J. Virol.73:3438-3442, 1999, and Clarke et al., J. Virol. 74:4831-4838, 2000;each incorporated herein by reference in its entirety for all purposes).

In more specific regard to the instant invention, a method for producingHPIV with a wt phenotype from cDNA was recently developed for recoveryof infectious, recombinant HPIV3 JS strain (see, e.g., Durbin et al.,Virology 235:323-332, 1997a; U.S. patent application Ser. No.09/083,793, filed May 22, 1998; U.S. Provisional Application Ser. No.60/047,575, filed May 23, 1997 (corresponding to InternationalPublication No. WO 98/53078), and U.S. Provisional Application Ser. No.60/059,385, filed Sep. 19, 1997, each incorporated herein by reference).In addition, these disclosures allow for genetic manipulation of viralcDNA clones to determine the genetic basis of phenotypic changes inbiological mutants, e.g., which mutations in the HPIV3 cp45 virusspecify its ts, ca and att phenotypes, and which gene(s) or genomesegment(s) of BPIV3 specify its attenuation phenotype. Additionally,these and related disclosures render it feasible to construct novel PIVvaccine candidates having a wide range of different mutations and toevaluate their level of attenuation, immunogenicity and phenotypicstability (see also, U.S. Provisional Patent Application Ser. No.60/143,134, filed by Bailly et al. on Jul. 9, 1999; and U.S. patentapplication Ser. No. 09/350,821, filed by Durbin et al. on Jul. 9, 1999;each incorporated herein by reference).

Thus, infectious wild type recombinant PIV3 (r)PIV3, as well as a numberof ts derivatives, have now been recovered from cDNA, and reversegenetics systems have been used to generate infectious virus bearingdefined attenuating mutations and to study the genetic basis ofattenuation of existing vaccine viruses. For example, the three aminoacid substitutions found in the L gene of cp45, singularly or incombination, have been found to specify the ts and attenuationphenotypes. Additional is and attenuating mutations are present in otherregions of the PIV3 cp45. In addition a chimeric PIV1 vaccine candidatehas been generated using the PIV3 cDNA rescue system by replacing thePIV3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3full-length cDNA that contains the three attenuating mutations in L. Therecombinant chimeric virus derived from this cDNA is designatedrPIV3-1.cp45L (Skiadopoulos et al., J. Virol. 72:1762-1768, 1998; Tao etal., J. Virol. 72:2955-2961, 1998; Tao et al., Vaccine 17:1100-1108,1999, incorporated herein by reference). rPIV3-1.cp45L was attenuated inhamsters and induced a high level of resistance to challenge with PIV1.A recombinant chimeric virus, designated rPIV3-1.cp45, has been producedthat contains 12 of the 15 cp45 mutations, i.e., excluding the mutationsthat occur in HN and F, and is highly attenuated in the upper and lowerrespiratory tract of hamsters (Skiadopoulos et al., Vaccine 18:503-510,1999a).

BPIV3, which is antigenically-related to HPIV3, offers an alternativeapproach to the development of a live attenuated virus vaccine forHPIV1, HPIV2, and HPIV3. The first vaccine used in humans, live vacciniavirus believed to be of bovine origin, was developed by Jenner almost200 years ago for the control of smallpox. During the ensuing twocenturies, vaccinia virus was successful in controlling this disease andplayed an essential role in the final eradication of smallpox. In this“Jennerian” approach to vaccine development, an antigenically-relatedanimal virus is used as a vaccine for humans. Animal viruses that arewell adapted to their natural host often do not replicate efficiently inhumans and hence are attenuated. At present, there is a lack of athorough understanding regarding the genetic basis for this form of hostrange restriction. Evolution of a virus in its mammalian or avian hostresults in significant divergence of nucleotide (nt) and amino acidsequences from that of the corresponding sequences in the related humanvirus. This divergent sequence, consisting of a large number of sequencedifferences, specifies the host range attenuation phenotype. Having anattenuation phenotype which is based on numerous sequence differences isa desirable property in a vaccine virus since it should contribute tothe stability of the attenuation phenotype of the animal virus followingits replication in humans.

The recently licensed quadrivalent rotavirus is an example of theJennerian approach to vaccine development in which a nonhuman rotavirusstrain, the rhesus rotavirus (RRV), was found to be attenuated in humansand protective against human serotype 3 to which it is antigenicallyhighly related (Kapikian et al., Adv. Exp. Med. Biol. 327:59-69, 1992).Since there was a need for a multivalent vaccine that would induceresistance to each of the four major human rotavirus serotypes, theJennerian approach was modified by constructing three reassortantviruses using conventional genetic techniques of gene reassortment intissue culture. Each single gene reassortant virus contained 10 RRVgenes plus a single human rotavirus gene that coded for the majorneutralization antigen (VP7) of serotype 1, 2, or 4. The intent was toprepare single gene substitution RRV reassortants with the attenuationcharacteristics of this simian virus and the neutralization specificityof human rotavirus serotype 1, 2, or 4. The quadrivalent vaccine basedon the host range restriction of the simian RRV in humans provided ahigh level of efficacy against human rotavirus infection in infants andyoung children (Perez-Schael et al., N. Engl. J. Med. 337:1181-1187,1997). However, the vaccine virus retains mild reactogenicity in olderseronegative infants lacking maternal antibody, therefore a secondgeneration Jennerian vaccine, based on the UK strain of bovinerotavirus, is being developed to replace the RRV vaccine (Clements-Mannet al., Vaccine 17:2715-2725, 1999).

The Jennerian approach also is being explored to develop vaccines forparainfluenza type 1 virus and for hepatitis A virus which areattenuated and immunogenic in non-human primates (Emerson et al., J.Infect. Dis. 173:592-597, 1996; Hurwitz et al., Vaccine 15:533-540,1997). The Jennerian approach was used for the development of a liveattenuated vaccine for influenza A virus but it failed to produce aconsistently attenuated vaccine for use in humans (Steinhoff et al., J.Infect. Dis. 163:1023-1028, 1991). As another example, reassortantviruses that contain two gene segments encoding the hemagglutinin andneuraminidase surface glycoproteins from a human influenza A virus andthe six remaining gene segments from an avian influenza A virus wereattenuated in humans (Clements et al., J. Clin. Microbiol. 27:219-222,1989; Murphy et al., J. Infect. Dis. 152:225-229, 1985; and Snyder etal., J. Clin. Microbiol. 23:852-857, 1986). This indicated that one ormore of the six gene segments of the avian virus attenuated theavian-human influenza A viruses for humans. The genetic determinants ofthis attenuation were mapped using reassortant viruses possessing asingle gene segment from an attenuating avian influenza A virus and theremaining genes from a human strain. It was shown that the nonstructural(NS), polymerase (PB1, PB2) and M genes contributed to the attenuationphenotype of avian influenza A viruses in humans (Clements et al., J.Clin. Microbiol. 30:655-662, 1992).

In another study, the severe host range restriction of bovinerespiratory syncytial virus (BRSV) for replication in chimpanzees wasonly slightly alleviated by replacement of the BRSV F and Gglycoproteins with their HRSV counterparts. This indicated that F and Gare involved in this host range restriction, but that one or moreadditional bovine RSV genes are also involved (Buchholz et al., J.Virol. 74:1187-1199, 2000). This illustrates that more than one gene cancontribute in unpredictable ways to the host range restriction phenotypeof a mammalian or avian virus in primates.

The instant invention provides a new basis for attenuating a wild typeor mutant parental virus for use as a vaccine against HPIV, in whichattenuation is based completely or in part on host range effects, whileat least one or more of the major neutralization and protectiveantigenic determinant(s) of the chimeric virus is homologous to thevirus against which the vaccine is directed. The HN and F proteins ofBPIV3 are each approximately 80% related by amino acid sequence to theircorresponding HPIV3 proteins (Suzu et al., Nucleic Acids Res.15:2945-2958, 1987, incorporated herein by reference) and 25% related byantigenic analysis (Coelingh et al., J. Virol. 64:3833-3843, 1990;Coelingh et al., J. Virol. 60:90-96, 1986; van Wyke Coelingh et al., J.Infect. Dis. 157:655-662, 1988, each incorporated herein by reference).Previous studies indicated that two strains of BPIV3, the Kansas (Ka)strain and the Shipping Fever (SF) prototype strain, were attenuated forthe upper and lower respiratory tract of rhesus monkeys, and one ofthese, the Ka strain, was attenuated in chimpanzees (van Wyke Coelinghet al., 1988, supra, incorporated herein by reference). Immunization ofnonhuman primates with the Ka virus induced antibodies reactive withHPIV3 and induced resistance to the replication of the human virus inthe upper and the lower respiratory tract of monkeys (id.) Subsequentevaluation of the Ka strain in humans indicated that the virus wassatisfactorily attenuated for seronegative infants, and it retained theattenuation phenotype following replication in filly susceptible infantsand children (Karron et al., 1996, supra; and Karron et al., 1995a,supra; each incorporated herein by reference). Its major advantagestherefore were that it was satisfactorily attenuated for fullysusceptible seronegative infants and children, and its attenuationphenotype was stable following replication in humans.

However, the level of serum hemagglutination-inhibiting antibodiesreactive with HPIV3 induced in seronegative vaccinees who received10^(5.0) tissue culture infectious dose₅₀(TCID)₅₀ of the Ka strain ofBPIV3 was 1:1 0.5, which was three-fold lower than similar vaccinees whoreceived a live attenuated HPIV3 vaccine (Karron et al., 1995a, supra;and Karron et al., 1995b, supra; each incorporated herein by reference).This lower level of antibodies to the human virus induced by BPIV3reflected in large part the antigenic divergence between HPIV3 and BPIV3(Karron et al., 1996, supra; and Karron et al., 1995a, supra; eachincorporated herein by reference). Studies to determine the efficacy ofthe Ka vaccine candidate against HPIV3 in humans have not beenperformed, but it is likely that this reduced level of antibodiesreactive with BPIV3 will be reflected in a reduced level of protectiveefficacy.

Although it is clear that BPIV3 has host range genes that restrictreplication in the respiratory tract of rhesus monkeys, chimpanzees andhumans, it remains unknown which of the bovine proteins or noncodingsequences contribute to this host range restriction of replication. Itis possible that any of the BPIV3 proteins or noncoding sequences mayconfer a host range phenotype. It is not possible to determine inadvance which genes or genome segments will confer an attenuationphenotype. This can only be accomplished by systematic substitution ofBPIV3 coding and non-coding sequences for their HPIV3 counterparts andby evaluation-of the recovered HPIV3/BPIV3 chimeric viruses inseronegative rhesus monkeys or humans.

Despite the numerous advances toward development of effective vaccineagents against PIV serotypes 1, 2, and 3, there remains a clear need inthe art for additional tools and methods to engineer safe and effectivevaccines to alleviate the serious health problems attributable to PIV,particularly illnesses among infants and children due to infection byHPIV. Among the remaining challenges in this context is the need foradditional tools to generate suitably attenuated, immunogenic andgenetically stable vaccine candidates for use in diverse clinicalsettings. To facilitate these goals, existing methods for identifyingand incorporating attenuating mutations into recombinant vaccine strainsmust be expanded. Furthermore, it is recognized that methods andcompositions for designing vaccines against human PIV can be implementedas well to design novel vaccine candidates for veterinary use.Surprisingly, the present invention fulfills these needs and providesadditional advantages as described hereinbelow.

SUMMARY OF THE INVENTION

The present invention provides human-bovine chimeric parainfluenzaviruses (PIVs) that are infectious and attenuated in humans and othermammals. In related aspects, the invention provides novel methods fordesigning and producing attenuated, human-bovine chimeric PIVs that areuseful in various compositions to generate a desired immune responseagainst PIV in a host susceptible to PIV infection. Included withinthese aspects of the invention are novel, isolated polynucleotidemolecules and vectors incorporating such molecules that comprise achimeric PIV genome or antigenome including a partial or complete humanor bovine PIV “background” genome or antigenome combined or integratedwith one or more heterologous gene(s) or genome segment(s) of adifferent PIV virus. Also provided within the invention are methods andcompositions incorporating human-bovine chimeric PIV for prophylaxis andtreatment of PIV infection.

The invention thus involves a method for developing live attenuated PIVvaccine candidates based on chimeras between HPIVs and BPIV3. Chimerasare generated using a cDNA-based virus recovery system. Recombinantviruses made from cDNA replicate independently and are propagated in thesame manner as if they were biologically-derived viruses. Chimerichuman-bovine PIV of the invention are recombinantly engineered toincorporate nucleotide sequences from both human and bovine PIV strainsto produce an infectious, chimeric virus or subviral particle. In thismanner, candidate vaccine viruses are recombinantly engineered to elicitan immune response against PIV in a mammalian host susceptible to PIVinfection, including humans and non-human primates. Human-bovinechimeric PIV according to the invention may be engeneered to elicit animmune response to a specific PIV, e.g., HPIV3, or a polyspecificresponse against multiple PIVs, e.g., HPIV1 and HPIV3. Additionalchimeric viruses can be designed in accordance with the teachings hereinwhich serve as vectors for antigens of non-PIV pathogens, for examplerespiratory syncytial virus (RSV) or measles virus.

Exemplary human-bovine chimeric PIV of the invention incorporate achimeric PIV genome or antigenome comprising both human and bovinepolynucleotide sequences, as well as a major nucleocapsid (N) protein, anucleocapsid phosphoprotein (P), and a large polymerase protein (L).Additional PIV proteins may be included in various combinations toprovide a range of infectious subviral particles, up to a complete viralparticle or a viral particle containing supernumerary proteins,antigenic determinants or other additional components.

Chimeric human-bovine PIV of the invention include a partial or complete“background” PIV genome or antigenome derived from or patterned after ahuman or bovine PIV strain or subgroup virus combined with one or moreheterologous gene(s) or genome segment(s) of a different PIV strain orsubgroup virus to form the human-bovine chimeric PIV genome orantigenome. In preferred aspects of the invention, chimeric PIVincorporate a partial or complete human PIV background genome orantigenome combined with one or more heterologous gene(s) or genomesegment(s) from a bovine PIV.

The partial or complete background genome or antigenome typically actsas a recipient backbone or vector into which are imported heterologousgenes or genome segments of the counterpart, human or bovine PIV.Heterologous genes or genome segments from the counterpart, human orbovine PIV represent “donor” genes or polynucleotides that are combinedwith, or substituted within, the background genome or antigenome toyield a human-bovine chimeric PIV that exhibits novel phenotypiccharacteristics compared to one or both of the contributing PIVs. Forexample, addition or substitution of heterologous genes or genomesegments within a selected recipient PIV strain may result in anincrease or decrease in attenuation, growth changes, alteredimmunogenicity, or other desired phenotypic changes as compared with acorresponding phenotype(s) of the unmodified recipient and/or donor.

Genes and genome segments that may be selected for use as heterologoussubstitutions or additions within human-bovine chimeric PIV of theinvention include genes or genome segments encoding a PIV N, P, C, D, V,M, F, SH (where appropriate), HN and/or L protein(s) or portion(s)thereof. In addition, genes and genome segments encoding non-PIVproteins, for example, an SH protein as found in mumps and SV5 viruses,may be incorporated within human-bovine PIV of the invention. Regulatoryregions, such as the extragenic 3′ leader or 5′ trailer regions, andgene-start, gene-end, intergenic regions, or 3′ or 5′ non-codingregions, are also useful as heterologous substitutions or additions.

Preferred human-bovine chimeric PIV vaccine candidates of the inventionbear one or more of the major antigenic determinants of HPIV3 in abackground which is attenuated by the substitution or addition of one ormore BPIV3 genes or genome segments. The major protective antigens ofPIVs are their HN and F glycoproteins, although other proteins can alsocontribute to a protective immune response. In certain embodiments, thebackground genome or antigenome is an HPIV genome or antigenome, e.g.,an HPIV3, HPIV2, or HPIV1 background genome or antigenome, to which isadded or into which is substituted one or more BPIV gene(s) or genomesegment(s), preferably from BPIV3. In one exemplary embodiment describedbelow, an ORF of the N gene of a BPIV3 is substituted for that of anHPIV. Alternatively, the background genome or antigenome may be a BPIVgenome or antigenome which is combined with one or more genes or genomesegments encoding a HPIV3, HPIV2, or HPIV1 glycoprotein, glycoproteindomain or other antigenic determinant.

In accordance with the methods of the invention, any BPIV gene or genomesegment, singly or in combination with one or more other BPIV genes, canbe combined with HPIV sequences to give rise to a human-bovine chimericPIV vaccine candidate. Any HPIV, including different strains of aparticular HPIV serotype, e.g., HPIV3 will be a reasonable acceptor forattenuating BPIV gene(s). In general, the HPIV3 gene(s) or genomesegment(s) selected for inclusion in a human-bovine chimeric PIV for useas a vaccine against human PIV will include one or more of the HPIVprotective antigens such as the HN or F glycoproteins.

In exemplary aspects of the invention, human-bovine chimeric PIV bearingone or more bovine gene(s) or genome segment(s) exhibits a high degreeof host range restriction, e.g., in the respiratory tract of mammalianmodels of human PIV infection such as non-human primates. In exemplaryembodiments a human PIV is attenuated by the addition or substitution ofone or more bovine gene(s) or genome segment(s) to a partial or completehuman, e.g., HPIV3, PIV background genome or antigenome. In one example,the HPIV3 N gene is substituted by the BPIV3 N gene to yield a novelhuman-bovine chimeric PIV vaccine candidate.

Preferably, the degree of host range restriction exhibited byhuman-bovine chimeric PIV vaccine candidates of the invention iscomparable to the degree of host range restriction exhibited by therespective BPIV parent or “donor” strain. Preferably, the restrictionshould have a true host range phenotype, i.e., it should be specific tothe host in question and should not restrict replication and vaccinepreparation in vitro in a suitable cell line. In addition, human-bovinechimeric PIV bearing one or more bovine gene(s) or genome segment(s)elicit a high level of resistance in hosts susceptible to PIV infection.Thus, the invention provides a new basis for attenuating a live virusvaccine against PIV, one which is based on host range effects due to theintroduction of one or more gene(s) or genome segment(s) from aheterologous PIV, e.g., between HPIV3 and BPIV3.

In related aspects of the invention, human-bovine chimeric PIVincorporates one or more heterologous gene(s) that encode an HPIV HNand/or F glycoprotein(s). Alternatively, the chimeric PIV mayincorporate one or more genome segment(s) encoding an ectodomain (andalternatively a cytoplasmic domain and/or transmembrane domain), orimmunogenic epitope of an HPIV HN and/or F glycoprotein(s). Theseimmunogenic proteins, domains and epitopes are particularly usefulwithin human-bovine chimeric PIV because they generate novel immuneresponses in an immunized host. In particular, the HN and F proteins,and immunogenic domains and epitopes therein, provide major protectiveantigens.

In certain embodiments of the invention, addition or substitution of oneor more immunogenic gene(s) or genome segment(s) from a human PIVsubgroup or strain to or within a bovine background, or recipient,genome or antigenome yields a recombinant, chimeric virus or subviralparticle capable of generating an immune response directed against thehuman donor virus, including one or more specific human PIV subgroups orstrains, while the bovine backbone confers an attenuated phenotypemaking the chimera a useful candidate for vaccine development. In oneexemplary embodiment, one or more human PIV glycoprotein genes, e.g., HNand/or F, are added to or substituted within a partial or completebovine genome or antigenome to yield an attenuated, infectioushuman-bovine chimera that elicits an anti-human PIV immune response in asusceptible host.

In alternate embodiments, human-bovine chimeric PIV additionallyincorporate a gene or genome segment encoding an immunogenic protein,protein domain or epitope from multiple human PIV strains, for exampletwo HN or F proteins or immunogenic portions thereof each from adifferent HPIV, e.g., HPIV1 or HPIV2. Alternatively, one glycoprotein orimmunogenic determinant may be provided from a first HPIV, and a secondglycoprotein or immunogenic determinant may be provided from a secondHPIV by substitution without the addition of an extra glycoprotein- ordeterminant-encoding polynucleotide to the genome or antigenome.Substitution or addition of BPIV glycoproteins and antigenicdeterminants may also be achieved by construction of a genome orantigenome that encodes a chimeric glycoprotein in the recombinant virusor subviral particle, for example having an immunogenic epitope,antigenic region or complete ectodomain of a first HPIV fused to acytoplasmic domain of a heterologous HPIV. For example, a heterologousgenome segment encoding a glycoprotein ectodomain from a HPIV1 or BPIV2HN or F glycoprotein may be joined with a genome segment encoding acorresponding HPIV3 HN or F glycoprotein cytoplasmic/endodomain in thebackground genome or antigenome.

In alternate embodiments a human-bovine chimeric PIV genome orantigenome may encode a substitute, extra, or chimeric glycoprotein orantigenic determinant thereof in the recombinant virus or subviralparticle, to yield a viral recombinant having both human and bovineglycoproteins, glycoprotein domains, or immunogenic epitopes. Forexample, a heterologous genome segment encoding a glycoproteinectodomain from a human PIV HN or F glycoprotein may be joined with agenome segment encoding a corresponding bovine HN or F glycoproteincytoplasmic/endodomain in the background genome or antigenome.Alternatively, the human PIV HN or F glycoprotein or parts thereof maybe joined with a genome segment encoding an HN or F glycoprotein orparts thereof from another PIV strain or serotype.

Thus, according to the methods of the invention, human-bovine chimericPIV may be constructed by substituting the heterologous gene or genomesegment for a counterpart gene or genome segment in a partial PIVbackground genome or antigenome. Alternatively, the heterologous gene orgenome segment may be added as a supernumerary gene or genome segment incombination with a complete (or partial if another gene or genomesegment is deleted) PIV background genome or antigenome. For example,two human PIV HN or F genes or genome segments can be included, one eachfrom HPIV2 and HPIV3.

Often, a heterologous gene or genome segment is added near an intergenicposition within a partial or complete PIV background genome orantigenome. Alternatively, the gene or genome segment can be placed inother noncoding regions of the genome, for example, within the 5′ or 3′noncoding regions or in other positions where noncoding nucleotidesoccur within the partial or complete genome or antigenome. In oneaspect, noncoding regulatory regions contain cis-acting signals requiredfor efficient replication, transcription, and translation, and thereforerepresent target sites for modification of these functions byintroducing a heterologous gene or genome segment or other mutation asdisclosed herein.

In more detailed aspects of the invention, attenuating mutations areintroduced into cis-acting regulatory regions to yield, e.g., (1) atissue specific attenuation (Gromeier et al., J. Virol. 73:958-964,1999; Zimmermann et al., J. Virol. 71:4145-4149, 1997), (2) increasedsensitivity to interferon (Zimmermann et al., 1997, supra), (3)temperature sensitivity (Whitehead et al., 1998a, supra), (4) a generalrestriction in level of replication (Men et al., J. Virol. 70:3930-3937,1996; Muster et al., Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991),and/or (5) host specific restriction of replication (Cahour et al.,Virology 207:68-76, 1995). These attenuating mutations can be achievedin various ways to produce an attenuated human-bovine chimeric PIV ofthe invention, for example by point mutations, swaps of sequencesbetween related viruses, or nucleotide deletions.

In yet additional alternative methods provided herein, a heterologousgene or genome segment may be added or substituted at a positioncorresponding to a wild-type gene order position of a counterpart geneor genome segment within the partial or complete PIV background genomeor antigenome. In other embodiments, the heterologous gene or genomesegment is added or substituted at a position that is morepromoter-proximal or promotor-distal compared to a wild-type gene orderposition of a counterpart gene or genome segment within the backgroundgenome or antigenome, to enhance or reduce expression, respectively, ofthe heterologous gene or genome segment.

In general aspects of the invention, bovine genes or genome segments maybe added to or substituted within a human PIV background to form anattenuated, human-bovine chimeric PIV. Alternatively, the chimera may becomprised of one or more human gene(s) or genome segment(s) added to orsubstituted within a bovine PIV background to form an attenuated PIVvaccine candidate. In this context, a chimeric PIV genome or antigenomeis formed of a partial or complete bovine PIV background genome orantigenome combined with a heterologous gene or genome segment from ahuman PIV. In preferred aspects, one or more bovine PIV gene(s) orgenome segment(s) is substituted for a counterpart gene(s) or genomesegment(s) within a human PIV background genome or antigenome. Inalternate embodiments, one or more human PIV glycoprotein genes, e.g.,HN and/or F or a genome segment encoding a cytoplasmic domain,transmembrane domain, ectodomain or immunogenic epitope of a human PIVglycoprotein gene is substituted for a counterpart gene or genomesegment within the bovine PIV background genome or antigenome. Forexample, both human PIV glycoprotein genes HN and F may be substitutedto replace counterpart HN and F glycoprotein genes in a bovine PIVbackground genome or antigenome.

In a parallel fashion, the chimeric human-bovine PIV of the inventioncan be readily designed as “vectors” to incorporate antigenicdeterminants from different pathogens, including more than one PIVstrain or group (e.g., both human PIV3 and human PIV1), respiratorysyncytial virus (RSV), measles and other pathogens (see, e.g., U.S.Provisional Patent Application Ser. No. 60/170,195, filed Dec. 10, 1999by Murphy et al., incorporated herein by reference).

In more detailed aspects of the invention, human-bovine chimeric PIV arecomprised of a partial or complete BPIV background genome or antigenomecombined with one or more heterologous gene(s) or genome segment(s) froma human PIV. Within these aspects, one or more of the HPIV glycoproteingenes HN and F, or one or more genome segments encoding a cytoplasmicdomain, transmembrane domain, ectodomain or immunogenic epitope of theHN and/or F genes, may be added to a BPIV background genome orantigenome or substituted for one or more counterpart genes or genomesegments within the BPIV background genome or antigenome to yield thechimeric construct. Often, both HPIV glycoprotein genes HN and F will besubstituted to replace counterpart HN and F glycoprotein genes in theBPIV background genome or antigenome, as exemplified by the recombinantchimeric virus rBPIV3-F_(H)HN_(H) described below. This is a desirableconstruct because it combines the antigenic determinants of the humanPIV with the host range restricting elements of the bovine PIV.

In combination with the host range phenotypic effects provided in thehuman-bovine chimeric PIV of the invention, it is often desirable toadjust the attenuation phenotype by introducing additional mutationsthat increase or decrease attenuation of the chimeric virus. Thus, inadditional aspects of the invention, attenuated, human-bovine chimericPIV are produced in which the chimeric genome or antigenome is furthermodified by introducing one or more attenuating mutations specifying anattenuating phenotype in the resultant virus or subviral particle. Thesecan include mutations in RNA regulatory sequences or in encodedproteins. These attenuating mutations may be generated de novo andtested for attenuating effects according to a rational designmutagenesis strategy. Alternatively, the attenuating mutations may beidentified in existing biologically derived mutant PIV and thereafterincorporated into a human-bovine chimeric PIV of the invention.

Introduction of attenuating and other desired phenotype-specifyingmutations into chimeric bovine-human PIV of the invention may beachieved by transferring a heterologous gene or genome segment, e.g., agene encoding an L protein or portion thereof, into a bovine or humanPIV background genome or antigenome. Alternatively, the mutation may bepresent in the selected background genome or antigenome, and theintroduced heterologous gene or genome segment may bear no mutations ormay bear one or more different mutations. Typically, the human or bovinebackground or “recipient” genome or antigenome is modified at one ormore sites corresponding to a site of mutation in a heterologous virus(e.g., a heterologous bovine or human PIV or a non-PIV negative strandedRNA virus) to contain or encode the same or a conservatively relatedmutation (e.g., a conservative amino acid substitution) as thatidentified in the donor virus (see, PCT/US00/09695 filed Apr. 12, 2000and its priority U.S. Provisional Patent Application Ser. No.60/129,006, filed Apr. 13, 1999, incorporated herein by reference). Inone exemplary embodiment, a bovine background or “recipient” genome orantigenome is modified at one or more sites corresponding to a site ofmutation in HPIV3 JS cp45, as enumerated below, to contain or encode thesame or a conservatively related mutation as that identified in the cp45“donor.”

Preferred mutant PIV strains for identifying and incorporatingattenuating mutations into bovine-human chimeric PIV of the inventioninclude cold passaged (cp), cold adapted (ca), host range restricted(hr), small plaque (sp), and/or temperature sensitive (ts) mutants, forexample the JS HPIV3 cp 45 mutant strain. In exemplary embodiments, oneor more attenuating mutations occur in the polymerase L protein, e.g.,at a position corresponding to Tyr₉₄₂, Leu₉₉₂, or Thr₁₅₅₈ of JS wildtype HPIV3. Alternatively, attenuating mutations in the N protein may beselected and incorporated in a human-bovine chimeric PIV, for examplewhich encode amino acid substitution(s) at a position corresponding toresidues Val₉₆ or Ser₃₈₉ of JS. Alternative or additional mutations mayencode amino acid substitution(s) in the C protein, e.g., at a positioncorresponding to Ile₉₆ of JS and in the M protein, e.g., at a positioncorresponding to Pro₁₉₉ (for example a Pro₁₉₉ to Thr mutation). Yetadditional mutations for adjusting attenuation of a human-bovinechimeric PIV of the invention are found in the F protein, e.g., at aposition corresponding to Ile₄₂₀ or Ala₄₅₀ of JS, and in the HN protein,e.g., at a position corresponding to residue Val₃₈₄ of JS.

Attenuating mutations from biologically derived PIV mutants forincorporation into human-bovine chimeric PIV of the invention alsoinclude mutations in noncoding portions of the PIV genome or antigenome,for example in a 3′ leader sequence. Exemplary mutations in this contextmay be engineered at a position in the 3′ leader of a recombinant virusat a position corresponding to nucleotide 23, 24, 28, or 45 of JS cp45.Yet additional exemplary mutations may be engineered in the N gene startsequence, for example by changing one or more nucleotides in the N genestart sequence, e.g., at a position corresponding to nucleotide 62 of JScp45.

From JS cp45 and other biologically derived PIV and non-PIV mutants, alarge “menu” of attenuating mutations is provided, each of whichmutations can be combined with any other mutation(s) for adjusting thelevel of attenuation in a recombinant PIV bearing a genome or antigenomethat is a chimera of human and bovine gene(s) or genome segment(s). Forexample, mutations within recombinant PIV of the invention include oneor more, and preferably two or more, mutations of JS cp45. Desiredhuman-bovine chimeric PIV of the invention selected for vaccine useoften have at least two and sometimes three or more attenuatingmutations to achieve a satisfactory level of attenuation for broadclinical use. Preferably, recombinant human-bovine chimeric PIVincorporate one or more attenuating mutation(s) stabilized by multiplenucleotide substitutions in a codon specifying the mutation.

Additional mutations which can be adopted or transferred to human-bovinechimeric PIV of the invention may be identified in non-PIV nonsegmentednegative standed RNA viruses and incorporated in PIV mutants of theinvention. This is readily accomplished by mapping the mutationidentified in a heterologous negative stranded RNA virus to acorresponding, homologous site in a recipient PIV genome or antigenomeand mutating the existing sequence in the recipient to the mutantgenotype (either by an identical or conservative mutation), as describedin PCT/US00/09695 filed Apr. 12, 2000 and its priority U.S. ProvisionalPatent Application Ser. No. 60/129,006, filed Apr. 13, 1999,incorporated herein by reference.

In addition to recombinant human-bovine chimeric PIV, the inventionprovides related cDNA clones, vectors and particles, each of whichincorporate HPIV and BPIV sequences and, optionally, one or more of theadditional, phenotype-specific mutations set forth herein. These areintroduced in selected combinations, e.g., into an isolatedpolynucleotide which is a recombinant cDNA genome or antigenome, toproduce a suitably attenuated, infectious virus or subviral particleupon expression, according to the methods described herein. Thisprocess, coupled with routine phenotypic evaluation, provideshuman-bovine chimeric PIV having such desired characteristics asattenuation, temperature sensitivity, altered immunogenicity,cold-adaptation, small plaque size, host range restriction, geneticstability, etc. In particular, vaccine candidates are selected which areattenuated and yet are sufficiently immunogenic to elicit a protectiveimmune response in the vaccinated mammalian host.

In yet additional aspects of the invention, human-bovine chimeric PIV,with or without additional mutations adopted, e.g., from a biologicallyderived attenuated mutant virus, are constructed to have additionalnucleotide modification(s) to yield a desired phenotypic, structural, orfunctional change. Typically, the selected nucleotide modification willspecify a phenotypic change, for example a change in growthcharacteristics, attenuation, temperature-sensitivity, cold-adaptation,plaque size, host range restriction, or immunogenicity. Structuralchanges in this context include introduction or ablation of restrictionsites into PIV encoding cDNAs for ease of manipulation andidentification.

In preferred embodiments, nucleotide changes within the genome orantigenome of a human-bovine chimeric PIV include modification of aviral gene by partial or complete deletion of the gene or reduction orablation (knock-out) of its expression. These modifications can beintroduced within the human or bovine background genome or antigenome,or may be introduced into the chimeric genome or antigenome byincorporation within the heterologous gene(s) or genome segment(s) addedor substituted therein. Target genes for mutation in this contextinclude any of the PIV genes, including the nucleocapsid protein N,phosphoprotein P, large polymerase subunit L, matrix protein M,hemagglutinin-neuraminidase protein HN, small hydrophobic SH protein,where applicable, fusion protein F, and the products of the C, D and Vopen reading frames (ORFs). To the extent that the recombinant virusremains viable and infectious, each of these proteins can be selectivelydeleted, substituted or rearranged, in whole or in part, alone or incombination with other desired modifications, to achieve novel deletionor knock out mutants. For example, one or more of the C, D, and/or Vgenes may be deleted in whole or in part, or its expression reduced orablated (e.g., by introduction of a stop codon, by a mutation in an RNAediting site, by a mutation that alters the amino acid specified by aninitiation codon, or by a frame shift mutation in the targeted ORF(s).In one embodiment, a mutation can be made in the editing site thatprevents editing and ablates expression of proteins whose mRNA isgenerated by RNA editing (Kato et al., EMBO J. 16:578-587, 1997a andSchneider et al., Virology 227:314-322, 1997, each incorporated hereinby reference). Alternatively, one or more of the C, D, and/or V ORF(s)can be deleted in whole or in part to alter the phenotype of theresultant recombinant clone to improve growth, attenuation,immunogenicity or other desired phenotypic characteristics (see, U.S.patent application Ser. No. 09/350,821, filed by Durbin et al. on Jul.9, 1999, incorporated herein by reference).

Alternative nucleotide modifications in human-bovine chimeric PIV of theinvention include a deletion, insertion, addition or rearrangement of acis-acting regulatory sequence for a selected gene in the recombinantgenome or antigenome. As with other such modifications described herein,these modifications can be introduced within the human or bovinebackground genome or antigenome, or may be introduced into the chimericgenome or antigenome by incorporation within the heterologous gene(s) orgenome segment(s) added or substituted therein. In one example, acis-acting regulatory sequence of one PIV gene is changed to correspondto a heterologous regulatory sequence, which may be a counterpartcis-acting regulatory sequence of the same gene in a different PIV, or acis-acting regulatory sequence of a different PIV gene. For example, agene end signal may be modified by conversion or substitution to a geneend signal of a different gene in the same PIV strain. In otherembodiments, the nucleotide modification may comprise an insertion,deletion, substitution, or rearrangement of a translational start sitewithin the recombinant genome or antigenome, e.g., to ablate analternative translational start site for a selected form of a protein.

In addition, a variety of other genetic alterations can be produced in ahuman-bovine chimeric PIV genome or antigenome, alone or together withone or more attenuating mutations adopted from a biologically derivedmutant PIV. For example, genes or genome segments from non-PIV sourcesmay be inserted in whole or in part. Alternatively, the order of genescan be changed, or a PIV genome promoter replaced with its antigenomecounterpart. Different or additional modifications in the recombinantgenome or antigenome can be made to facilitate manipulations, such asthe insertion of unique restriction sites in various non-coding regionsor elsewhere. Nontranslated gene sequences can be removed to increasecapacity for inserting foreign sequences.

In yet additional aspects, polynucleotide molecules or vectors encodingthe human-bovine chimeric PIV genome or antigenome can be modified toencode non-PIV sequences, e.g., a cytokine, a T-helper epitope, arestriction site marker, or a protein or immunogenic epitope of amicrobial pathogen (e.g., virus, bacterium, parasite, or fungus) capableof eliciting a protective immune response in an intended host. In onesuch embodiment, human-bovine chimeric PIV are constructed thatincorporate a gene or genome segment from a respiratory syncytial virus(RSV), for example a gene encoding an antigenic protein (e.g., an F or Gprotein), immunogenic domain or epitope of RSV.

In related aspects of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating a PIV-encoding cDNA) andmethods are provided for producing an isolated infectious human-bovinechimeric PIV. Included within these aspects of the invention are novel,isolated polynucleotide molecules and vectors incorporating suchmolecules that comprise a human-bovine chimeric PIV genome orantigenome. Also provided is the same or different expression vectorcomprising one or more isolated polynucleotide molecules encoding N, P,and L proteins. These proteins also can be expressed directly from thegenome or antigenome cDNA. The vector(s) is/are preferably expressed orcoexpressed in a cell or cell-free lysate, thereby producing aninfectious human-bovine chimeric PIV viral particle or subviralparticle.

The above methods and compositions for producing human-bovine chimericPIV yield infectious viral or subviral particles, or derivativesthereof. A recombinant infectious virus is comparable to the authenticPIV virus particle and is infectious as is. It can directly infect freshcells. An infectious subviral particle typically is a subcomponent ofthe virus particle which can initiate an infection under appropriateconditions. For example, a nucleocapsid containing the genomic orantigenomic RNA and the N, P, and L proteins is an example of a subviralparticle which can initiate an infection if introduced into thecytoplasm of cells. Subviral particles provided within the inventioninclude viral particles which lack one or more protein(s), proteinsegment(s), or other viral component(s) not essential for infectivity.

In other embodiments the invention provides a cell or cell-free lysatecontaining an expression vector which comprises an isolatedpolynucleotide molecule comprising a human-bovine chimeric PIV genome orantigenome as described above, and an expression vector (the same ordifferent vector) which comprises one or more isolated polynucleotidemolecules encoding the N, P, and L proteins of PIV. One or more of theseproteins also can be expressed from the genome or antigenome cDNA. Uponexpression the genome or antigenome and N, P and L combine to produce aninfectious human-bovine chimeric PIV virus or subviral particle.

The human-bovine chimeric PIVs of the invention are useful in variouscompositions to generate a desired immune response against PIV in a hostsusceptible to PIV infection. Human-bovine chimeric PIV recombinants arecapable of eliciting a protective immune response in an infectedmammalian host, yet are sufficiently attenuated so as not to causeunacceptable symptoms of severe respiratory disease in the immunizedhost. In addition, the human-bovine chimeric PIV recombinants shouldreplicate with sufficient efficiency in vitro to make vaccinepreparation feasible. The attenuated virus or subviral particle may bepresent in a cell culture supernatant, isolated from the culture, orpartially or completely purified. The virus may also be lyophilized, andcan be combined with a variety of other components for storage ordelivery to a host, as desired.

The invention further provides novel vaccines comprising aphysiologically acceptable carrier and/or adjuvant and an isolatedattenuated human-bovine chimeric PIV virus or subviral particle. Inpreferred embodiments, the vaccine is comprised of a human-bovinechimeric PIV having at least one, and preferably two or more additionalmutations or other nucleotide modifications as described above toachieve a suitable balance of attenuation and immunogenicity. Thevaccine can be formulated in a dose of 10³ to 10⁷ PFU of attenuatedvirus. The vaccine may comprise attenuated human-bovine chimeric PIVthat elicits an immune response against a single PIV strain or againstmultiple PIV strains or groups. In this regard, human-bovine chimericPIV can be combined in vaccine formulations with other PIV vaccinestrains, or with other viral vaccine viruses such as RSV.

In related aspects, the invention provides a method for stimulating theimmune system to elicit an immune response against PIV in a mammaliansubject. The method comprises administering a formulation of animmunologically sufficient amount of a human-bovine chimeric PIV in aphysiologically acceptable carrier and/or adjuvant. In one embodiment,the immunogenic composition is a vaccine comprised of a human-bovinechimeric PIV having at least one, and preferably two or more attenuatingmutations or other nucleotide modifications specifying a desiredphenotype as described above. The vaccine can be formulated in a dose of10³ to 10⁷ PFU of attenuated virus. The vaccine may comprise attenuatedhuman-bovine chimeric PIV virus that elicits an immune response againsta single PIV, against multiple PIVs, e.g., HPIV1 and HPIV3, or againstone or more PIV(s) and a non-PIV pathogen such as RSV. In this context,human-bovine chimeric PIV can elicit a monospecific immune response or apolyspecific immune response against multiple PIVs, or against one ormore PIV(s) and a non-PIV pathogen such as RSV. Alternatively,human-bovine chimeric PIV having different immunogenic characteristicscan be combined in a vaccine mixture or administered separately in acoordinated treatment protocol to elicit more effective protectionagainst one PIV, against multiple PIVs, or against one or more PIV(s)and a non-PIV pathogen such as RSV. Preferably the immunogeniccomposition is administered to the upper respiratory tract, e.g., byspray, droplet or aerosol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates cloning of the N coding region of bovine PIV strainsKa or SF into HPIV3. In FIG. 1A-C, the BPIV3 N open reading frame (ORF)replaces its corresponding HPIV3 sequence in the full-length rJSantigenomic cDNA (Durbin et al., 1997a, supra). BPIV3 Ka and SF N geneswere first amplified by RT-PCR using standard molecular biologicaltechniques from virion RNA and subcloned as 1.9 kb fragments intopBluescript to give pBS-KaN or pBS-SFN, respectively. The HPIV3 rJS Ngene was subcloned as a 1.9 kb Mlul/EcoRI fragment into pUC 119 from aplasmid containing the 5′ half of the rJS HPIV3 antigenome (Durbin etal., 1997a, supra; U.S. patent application Ser. No. 09/083,793, filedMay 22, 1998; U.S. Provisional Application No. 60/047,575, filed May 23,1997 (corresponding to International Publication No. WO 98/53078), andU.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997, eachincorporated herein by reference) to give pUC119JSN. Each N gene wasmodified by site-directed mutagenesis to place an Ncol and AflII site atthe translational start and stop sites, respectively. The Ka and SF Ngenes are identical in the translational start and stop site regionsand, therefore, identical mutagenesis reactions were performed on bothBPIV3 N genes as depicted in LA. FIG. 1B—Following AflII/Ncol digestion,a 1.5 kb fragment from pBS-KaN or pBS-SFN representing the BPIV3 Ncoding region was introduced into the Ncol/AflII window of the HPIV3 Nsubclone pUC119JSN-Ncol/AflII as a replacement for its HPIV3counterpart. FIG. 1C—Each chimeric subclone was then subjected tosite-directed mutagenesis to restore the sequence present in HPIV3 rJSbefore the translation start codon or after the stop codon and BPIV3coding sequence immediately after the start codon and before the stopcodon. This yielded pUC119B/HKaN and pUC119B/HSFN, which were used toimport the BPIV3 N gene into the HPIV3 cDNA clone as shown in FIG. 2.FIG. 1, Panel A, CAAAAATGTTG (SEQ ID NO. 10); GCAACTAATCGA (SEQ ID NO.11); TAACCATGGTGA (SEQ ID NO. 12); GCACTTAAGCAC (SEQ ID NO. 13). FIG. 1,Panel C, TAACCATGGTGA (SEQ ID NO. 12); GCACTTAAGCAC (SEQ ID NO. 13);CAAAAATGTTGA (SEQ ID NO. 14); GCAACTAGTCGA (SEQ ID NO. 15).

FIG. 2 illustrates insertion of the HPIV3/BPIV3 (strain Ka or SF)chimeric N gene into the HPIV3 antigenomic cDNA. In FIG. 2A, the BPIV3 NORF of Ka or SF flanked by HPIV3 sequence was subcloned as an Mlul/EcoRIfragment from pUC119B/HKaN or pUC119B/HSFN and inserted into pLeft+2G(Durbin et al., 1997a, supra). The pLeft+2G plasmid contains the 5′ halfof the HPIV3 rJS antigenome from nt 1-7437 (genome sense) behind a T7promoter. The location of two G residues that were inserted between T7promoter and HPIV3 sequence to improve transcription is indicated by anasterisk. FIG. 2B—An Xhol/NgoMl fragment of pRight (Durbin et al.,1997a, supra; U.S. patent application Ser. No. 09/083,793, filed May 22,1998; U.S. Provisional Application No. 60/047,575, filed May 23, 1997(corresponding to International Publication No. WO 98/53078), and U.S.Provisional Application No. 60/059,385, filed Sep. 19, 1997, eachincorporated herein by reference) containing the 3′ end of the HPIV3antigenome-flanked by the hepatitis delta virus ribozyme and T7terminator was cloned into the Xhol/NgoMl window of the modified pLeftplasmid resulting in plasmids pB/HPIV3KaN and pB/HPIV3SFN. Each of thesechimeric constructs contains the complete positive sense sequence of theHPIV3 antigenomic RNA except for the N coding region which has beenreplaced by its BPIV3 Ka or SF counterpart.

FIG. 3 provides nucleotide sequences of HPIV3, BPIV3 and chimericviruses of the invention around N translation start (A) and stop (B)codons. The position of the individual ORFs is described in therespective Genbank reports (#AF178654 for BPIV3 Ka, #AF178655 for BPIV3SF and #Z11515) and included herein by reference. The sequences(positive-sense) flanking the translational start (A) and stop (B)codons (each underlined) in the N gene are shown for the parentalrecombinant HPIV3 JS (rJS), the parental biologically-derived BPIV3 Kaand SF viruses (Ka and SF), and the chimeric cKa and cSF viruses.Host-specific residues in the cKa and cSF virus sequences and theircounterparts in rJS (before the start codon and after the stop codon)and SF or Ka (start codon through stop codon, inclusive) are in boldfacetype. Plaque-purified chimeric virus was amplified by RT-PCR from virionRNA and sequenced using the Taq Dye Deoxy Terminator Cycle kit (ABI,Foster City, Calif.). This confirmed that the predicted sequences werepresent in each chimeric virus. FIG. 3A. rJS,GGAACTCTATAATTTCAAAAATGTTGAGCCTATTTGATAC (SEQ ID NO. 16). FIG. 3A. cKaand cSF, GGAACTCTATAATTTCAAAAATGTTGAGTCTATTCGACAC (SEQ ID NO. 17). FIG.3A. Ka and SF, GAAATCCTAAGACTGTAATCATGTTGAGTCTATTCGACAC (SEQ ID NO. 18).FIG. 3B. rJS, TTAACGCATTTGGAAGCAACTAATCGAATCAACATTTTAA (SEQ ID NO. 19).FIG. 3B. cKa and cSF, TCAGTGCATTCGGAAGCAACTAGTCGAATCAACATTTTAA (SEQ IDNO. 20). FIG. 3B. Ka and SF, TCAGTGCATTCGGAAGCAACTAGTCACAAAGAGATGACCA(SEQ ID NO. 21).

FIG. 4 details the structure of the BPIV3/HPIV3 chimeric viruses of theinvention, and their confirmation by TaqI digestion of RT-PCR productsgenerated from virus RNA. In FIG. 4A the genomes of the chimeric cKa andcSF viruses are shown schematically (not to scale) relative to that ofHPIV3 and BPIV3 parent viruses. Ka- and SF-specific regions areindicated by light and dark shading respectively. Arrows above the rJSgenome indicate the locations of primers used for RT-PCR amplificationof chimeric and parent viruses for the purposes of diagnostic TaqIdigestion. These primers were directed to regions conserved betweenHPIV3 and BPIV3 so that they could be used for the amplification ofHPIV3, BPIV3 and chimeric BPIV3/HPIV3 viruses. In FIG. 4B the expectedsizes of TaqI digestion products for each virus are shown for a 1898-bpPCR product amplified from RNA with the primer pair illustrated in FIG.4A. This PCR product is illustrated at the top in FIG. 4B, and the N ORFis indicated as a filled rectangle. TaqI fragments unique to each virusand which therefore serve in virus identification are indicated with anasterisk. FIG. 4C provides TaqI profiles of PCR products containing thePIV3 N coding region of chimeric cKa (left) or cSF (right) flanked bythose of the HPIV3 and BPIV3 parent viruses. Unique TaqI fragmentsdiagnostic of virus identity and corresponding to those identified in(4B) are indicated with an asterisk. Calculated lengths (bp) of DNA gelbands are indicated.

FIG. 5 provides multicycle growth curves of parental and chimericviruses in MDBK (A) or LLC-MK2 (B) cells. Monolayers of bovine MDBK (A)or simian LLC-MK2 (B) cells in wells (9.6 cm² each) of a 6 well platewere infected individually at a multiplicity of infection of 0.01 withthe indicated parental or chimeric virus. Three replicate infectionswere performed for each virus. Samples were taken at the indicated timepoints, stored at −70° C., and titered by TCID₅₀ assay in parallel.Growth curves are constructed using the average of 3 replicate samplesat each time point. The lower limit of virus detectability was10^(1.5)TCID₅₀/ml, which is indicated by a dotted line.

FIGS. 6A-6G set forth the complete positive sense nucleotide sequence(SEQ ID NO. 22) of the bovine PIV3 Ka strain.

FIGS. 7A-7G set forth the complete positive sense nucleotide sequence(SEQ ID NO. 23) of the bovine PIV3 SF stain.

FIG. 8A provides a schematic depiction of the genomes of chimericrHPIV3-F_(B)HN_(B) and rBPIV3-F_(H)HN_(H) viruses, and of their parentviruses, rHPIV3 JS and BPIV3 Ka (not to scale). The F and HN genes wereexchanged in a single restriction fragment between rHPIV3 and rBPIV3using SgrAI and BsiWI sites that had been introduced in front of the Mand HN gene end sequences, respectively.

FIG. 8B depicts assembly of an antigenomic cDNA for BPIV3 Ka. A fulllength cDNA was constructed to encode the complete antigenomic sequenceof BPIV3 Ka (GenBank accession #AF178654). The cDNA was assembled fromsubclones derived from reverse transcription (RT) of viral (v)RNA andpolymerase chain reaction (PCR) amplification. Multiple subclones of theantigenome were sequenced, and only clones matching the consensussequence of BPIV3 Ka were used for assembly of the full length clone,with the exception of nt 21 and nt 23, which differ from the publishedsequence but occur with similar frequency in the virus population.

FIG. 8C illustrates features of parental and chimeric bovine-human PIVgenomes. The genomes of the chimeric rHPIV3 F_(B)HN_(B) and rBPIV3F_(H)HN_(H) viruses and those of their parent viruses rHPIV3 JS andBPIV3 Ka are shown schematically (not to scale). Two unique restrictionenzyme recognition sites, SgrAI and BsiWI, were introduced near the Mand HN gene ends, respectively. The recombinant HPIV3 and BPIV3 virusesbearing these introduced restriction sites were designeated rHPIV3s andrBPIV3s as indicated in Fugure 8C2. Glycoprotein genes were exchangedbetween rHPIV3 JS and rBPIV3 Ka. The nucleotide sequence that wasmutagenized is shown below each cDNA construct, with the position of thefirst nucleotide of each sequence indicated. The introduced SgrAI andBsiWI restriction sites are underlined and nucleotides that differbetween HPIV3 and BPIV3 and thus identify the origin of the gene insertsare depicted in bold print. FIG. 8C, Panel 1, rHPIV 3 JS, TCCACCGGTGCA(SEQ ID NO. 4), TAGACAAAAGGG (SEQ ID NO. 24). FIG. 8C, Panel 1. rBPIV3Ka, TCCAACATTGCA (SEQ ID NO. 2); AAGATATAAAGA (SEQ ID NO. 25). FIG. 8C,Panel 2 rHPIV3s, CGCACCGGTGTA (SEQ ID NO. 5); TAGACGTACGGG (SEQ ID NO.26). FIG. 8C, Panel 2. rBPIV3s, TCCACCGGTGCA (SEQ ID NO. 3);AAGACGTACGGA (SEQ ID NO. 27). FIG. 8C, Panel 3. rHPIV3 F_(B)HN_(B),CGCACCGGTGCA (SEQ ID NO. 28); AAGACGTACGGG (SEQ ID NO. 29). FIG. 8C,Panel 3. rBPIV3 F_(H)HN_(H), AAGACGTACGGG (SEQ ID NO. 30); TAGACGTACGGA(SEQ ID NO. 31).

FIG. 9 provides a confirmation of the identity of recombinant viruses byRT-PCR of viral RNA and Eco RI digestion. RT-PCR products of viral RNAwere prepared with a primer pair that recognized conserved regions oneither side of the F and HN genes in both BPIV3 and HPIV3. Digestionwith Eco RI resulted in a unique pattern of restriction fragments foreach of the four viruses. In the schematic diagram on the left,horizontal lines symbolize the amplified viral sequences and verticalbars show the positions of Eco RI sites. The expected size of eachrestriction fragment is indicated above the line. The numbers below eachline correspond to the sequence position in the antigenomic RNA of BPIV3Ka, HPIV3 JS (GenBank accession #AF178654 and Z11575), or of theindicated chimeric derivative. On the right, a 1% agarose gel of the EcoRI digestion of PCR products is shown, confirming the identity ofparental and chimeric viruses. The asterisks indicate gel bands thatcontain two restriction fragments that comigrate due to close similarityin size.

FIG. 10 depicts multicycle replication of chimeric and parental virusesin simian LLC-MK2 cells. Multicycle replication (the input inoculum hadan MOI of 0.01) of the three chimeric viruses rHPIV3-F_(B)HN_(B),rBPIV3-F_(H)HN_(H) and rHPIV3-N_(B) (also referred to as cKa) iscompared with the replication of their parental viruses BPIV3 Ka andrBPIV3. The virus titers are shown as mean log₁₀ TCID₅₀/ml±standarderror of triplicate samples. The lower limit of detection of this assayis 10 TCID₅₀, as indicated by the dotted horizontal line.

FIG. 11 documents mean titers of chimeric and parental viruses innasopharyngeal swabs of infected rhesus monkeys over the course ofinfection. Virus titers are shown as mean TCID₅₀/ml in LLC-MK2cells±standard error for groups of 4 or 6 monkeys infected with the samevirus. This illustrates the same experiment as shown in Table 3. Inpanel A, mean titers of rHPIV3-F_(B)HN_(B) are compared to rHPIV3 andBPIV3 Ka titers. In panel B, mean rBPIV3-F_(H)HN_(H) titers are comparedto those of BPIV3 Ka and rHPIV3, which, for the last two viruses, arethe same values in panel A but are presented separately to facilitatecomparison. Day 5 titers were excluded from the FIGS. because they weremuch lower than day 4 and day 6 titers, most likely due to technicalproblems during the sample collection.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides recombinant parainfluenza virus (PIV)cloned as a chimera of human and bovine PIV genomic or antigenomicsequences to yield a human-bovine chimeric PIV. The chimericconstruction of human-bovine PIV yields a viral particle or subviralparticle that is infectious in mammals, particularly humans, and usefulfor generating immunogenic compositions for clinical or veterinary use.Also provided within the invention are novel methods and compositionsfor designing and producing attenuated, human-bovine chimeric PIV, aswell as methods and compositions for the prophylaxis and treatment ofPIV infection.

Chimeric human-bovine PIV of the invention are recombinantly engineeredto incorporate nucleotide sequences from both human and bovine PIVstrains to produce an infectious, chimeric virus or subviral particle.In this manner, candidate vaccine viruses are recombinantly engineeredto elicit an immune response against PIV in a mammalian host susceptibleto PIV infection, including humans and non-human primates. Human-bovinechimeric PIV according to the invention may elicit an immune response toa specific PIV, e.g., HPIV3, or a polyspecific response against multiplePIVs, e.g., HPIV1 and HPIV3.

Exemplary human-bovine chimeric PIV of the invention incorporate achimeric PIV genome or antigenome comprising both human and bovinepolynucleotide sequences, as well as a major nucleocapsid (N) protein, anucleocapsid phosphoprotein (P), and a large polymerase protein (L).Additional PIV proteins may be included in various combinations toprovide a range of infectious subviral particles, up to a complete viralparticle or a viral particle containing supernumerary proteins,antigenic determinants or other additional components.

Chimeric human-bovine PIV of the invention include a partial or complete“background” PIV genome or antigenome derived from or patterned after ahuman or bovine PIV strain or serotype virus combined with one or moreheterologous gene(s) or genome segment(s) of a different PIV strain orserotype virus to form the human-bovine chimeric PIV genome orantigenome. In certain aspects of the invention, chimeric PIVincorporate a partial or complete human PIV (HPIV) background genome orantigenome combined with one or more heterologous gene(s) or genomesegment(s) from a bovine PIV. In alternate aspects of the invention,chimeric PIV incorporate a partial or complete bovine PIV (BPIV)background genome or antigenome combined with one or more heterologousgene(s) or genome segment(s) from a human PIV.

The partial or complete background genome or antigenome typically actsas a recipient backbone or vector into which are imported heterologousgenes or genome segments of the counterpart, human or bovine PIV.Heterologous genes or genome segments from the counterpart, human orbovine PIV represent “donor” genes or polynucleotides that are combinedwith, or substituted within, the background genome or antigenome toyield a human-bovine chimeric PIV that exhibits novel phenotypiccharacteristics compared to one or both of the contributing PIVs. Forexample, addition or substitution of heterologous genes or genomesegments within a selected recipient PIV strain may result in anincrease or decrease in attenuation, growth changes, alteredimmunogenicity, or other desired phenotypic changes as compared with acorresponding phenotype(s) of the unmodified recipient and/or donor.Genes and genome segments that may be selected for use as heterologousinserts or additions within human-bovine chimeric PIV of the inventioninclude genes or genome segments encoding a PIV N, P, C, D, V, M, SH,where applicable, F, HN and/or L protein(s) or portion(s) thereof.Regulatory regions, such as the extragenic leader or trailer orintergenic regions, are also useful as heterologous inserts oradditions.

The heterologous gene(s) or genome segment(s) may be added orsubstituted at a position corresponding to a wild-type gene orderposition of the counterpart gene(s) or genome segment(s) within thepartial or complete PIV background genome or antigenome, whichcounterpart gene or genome segment is thereby replaced or displaced(e.g., to a more promotor-distal position). In yet additionalembodiments, the heterologous gene or genome segment is added orsubstituted at a position that is more promoter-proximal orpromotor-distal compared to a wild-type gene order position of thecounterpart gene or genome segment within the background genome orantigenome, which enhances or reduces, respectively, expression of theheterologous gene or genome segment.

The introduction of heterologous immunogenic proteins, domains andepitopes to produce human-bovine chimeric PIV is particularly useful togenerate novel immune responses in an immunized host. Addition orsubstitution of an immunogenic gene or genome segment from one, donorPIV within a recipient genome or antigenome of a different PIV cangenerate an immune response directed against the donor subgroup orstrain, the recipient subgroup or strain, or against both the donor andrecipient subgroup or strain. To achieve this purpose, human-bovinechimeric PIV may also be constructed that express a chimeric protein,e.g., an immunogenic glycoprotein having a cytoplasmic tail and/ortransmembrane domain specific to one PIV fused to an ectodomain of adifferent PIV to provide, e.g., a human-bovine fusion protein, or afusion protein incorporating domains from two different human PIVs. In apreferred embodiment, a human-bovine chimeric PIV genome or antigenomeencodes a chimeric glycoprotein in the recombinant virus or subviralparticle having both human and bovine glycoprotein domains orimmunogenic epitopes. For example, a heterologous genome segmentencoding a glycoprotein ectodomain from a human PIV HN or F glycoproteinmay be joined with a polynucleotide sequence (i.e., a genome segment)encoding the corresponding bovine HN or F glycoprotein cytoplasmic andtransmembrane domains to form the human-bovine chimeric PIV genome orantigenome.

In other embodiments, human-bovine chimeric PIV useful in a vaccineformulation can be conveniently modified to accommodate antigenic driftin circulating virus. Typically the modification will be in the HNand/or F proteins. This might involve the introduction of one or morepoint mutations; it might also involve an entire HN or F gene, or agenome segment encoding a particular immunogenic region thereof, fromone PIV strain or group is incorporated into a chimeric PIV genome orantigenome cDNA by replacement of a corresponding region in a recipientclone of a different PIV strain or group, or by adding one or morecopies of the gene, such that multiple antigenic forms are represented.Progeny virus produced from the modified PIV clone can then be used invaccination protocols against emerging PIV strains.

Replacement of a human PIV coding sequence or non-coding sequence (e.g.,a promoter, gene-end, gene-start, intergenic or other cis-actingelement) with a heterologous counterpart yields chimeric PIV having avariety of possible attenuating and other phenotypic effects. Inparticular, host range and other desired effects arise from substitutinga bovine or murine PIV (MPIV) protein, protein domain, gene or genomesegment imported within a human PIV background, wherein the bovine ormurine gene does not function efficiently in a human cell, e.g., fromincompatibility of the g heterologous sequence or protein with abiologically interactive human PIV sequence or protein (i.e., a sequenceor protein that ordinarily cooperates with the substituted sequence orprotein for viral transcription, translation, assembly, etc.) or, moretypically in a host range restriction, with a cellular protein or someother aspect of the cellular milieu which is different between thepermissive and less permissive host. In exemplary embodiments, bovinePIV sequences are selected for introduction into human PIV based onknown aspects of bovine and human PIV structure and function.

HPIV3 is a member of the Respirovirus genus of the Paramyxoviridaefamily in the order Mononegavirales (Collins et al., 1996, supra). HPIV3is the best characterized of the HPIVs and represents the prototypeHPIV. Its genome is a single strand of negative-sense RNA 15462nucleotides (nt) in length (Galinski et al., Virology 165:499-510, 1988;and Stokes et al., Virus Res. 25:91-103, 1992; each incorporated hereinby reference). At least eight proteins are encoded by the PIV3 genome:the nucleocapsid protein N, the phosphoprotein P, the C and D proteinsof unknown functions, the matrix protein M, the fusion glycoprotein F,the hemagglutinin-neuraminidase glycoprotein HN, and the largepolymerase protein L (Collins et al., 1996, supra). A protein containingthe V ORF in the P gene might also be produced (Durbin et al., Virology261:319-333,1999)

The M, HN, and F proteins are envelope-associated, and the latter twoare surface glycoproteins which, as is the case with each PIV, are themajor neutralization and protective antigens (Collins et al., 1996,supra). The significant sequence divergence between comparable PIV HN orF proteins among the PIVs is thought to be the basis for the typespecificity of the protective immunity (Collins et al., 1996, supra;Cook et al., Amer. Jour. Hyg. 77:150-159, 1963; Ray et al., J. Infect.Dis. 162:746-749, 1990; each incorporated herein by reference).

The HPIV3 genes are each transcribed as a single mRNA that encodes asingle protein, with the exception of the P mRNA which contains fourORFs, namely P, C, D and V (Galinski et al., Virology 186:543-550, 1992;and Spriggs et al., J. Gen. Virol. 67:2705-2719, 1986; each incorporatedherein by reference). The P and C proteins are translated from separate,overlapping ORFs in the mRNA. Whereas all paramyxoviruses encode a Pprotein, only members of the genus Respirovirus and Morbillivirus encodea C protein. Individual viruses vary in the number of proteins expressedfrom the C ORF and in its importance in replication of the virus invitro and in vivo. Sendai virus (SeV) expresses four independentlyinitiated proteins from the C ORF: C′, C, Y1, and Y2, whosetranslational start sites appear in that order in the mRNA (Cumin, etal., Enzyme 44:244-249,1990; Lamb et al., in The Paramyxoviruses. D.Kingsbury, ed., pp. 181-214, Plenum Press, New York, 1991; incorporatedherein by reference), whereas HPIV3 and measles virus (MeV) express onlya single C protein (Bellini et al., J. Virol. 53:908-919, 1985; Sanchezet al., Virology 147:177-86, 1985; and Spriggs et al., 1986, supra; eachincorporated herein by reference).

The PIV3 D protein is a fusion protein of the P and D ORFs, and isexpressed from the P gene by the process of co-transcriptional RNAediting in which two nontemplated G residues are added to the P mRNA atthe RNA editing site (Galinski et al., 1992, supra; and Pelet et al.,EMBO J. 10:443-448, 1991; each incorporated herein by reference). BPIV3,the only other paramyxovirus which expresses a D protein, uses RNAediting to express this protein as well as a second protein, the Vprotein.

Nearly all members of the genera Respirovirus, Rubulavirus, andMorbillivirus express a V protein. The one member which clearly does notis HPIV1, which lacks an intact V ORF (Matsuoka et al., J. Virol.65:3406-3410, 1991, incorporated herein by reference). The V ORF ischaracterized by the presence of a cysteine-rich domain that is-highlyconserved (Cattaneo et al., Cell 56:759-764, 1989; Park et al., J.Virol. 66:7033-7039, 1992; Thomas et al., Cell 54:891-902, 1988; andVidal et al., J. Virol. 64:239-246, 1990; each incorporated herein byreference). The V ORF is maintained in each of the HPIV3 virusessequenced to date suggesting that this ORF is expressed and retainsfunction for this virus (Galinski et al., Virology 155:46-60, 1986;Spriggs et al., 1986, supra; and Stokes et al., 1992, supra;incorporated herein by reference).

The BPIV3 V protein is expressed when one nontemplated G residue isadded at the RNA editing site (Pelet et al., 1991, supra; incorporatedherein by reference). However, in the case of HPIV3, two translationstop codons lie between the editing site and the V ORF, and it is notclear whether HPIV3 represents another example in which this ORF is notexpressed, or whether it is expressed by some other mechanism. Onepossibility is that HPIV3 editing also occurs at a second, downstreamsite in the P gene, although this did not appear to occur in cellculture (Galinski et al., 1992, supra). Alternatively, it might be thatribosomes gain access to the V ORF by ribosomal frameshifting. Thiswould be comparable to the situation with the P locus of MV. MVexpresses C, P, and V proteins, but also expresses a novel R proteinwhich is synthesized by frameshifting from the P ORF to the V ORF(Liston et al., J. Virol. 69:6742-6750, 1995, incorporated herein byreference). Genetic evidence suggests that the V ORF of HPIV3 isfunctional (Durbin et al., 1999, supra).

Although the means by which HPIV3 expresses its V protein is unclear,the extreme conservation of the its V ORF in different strains suggeststhat it is indeed expressed. The function of the V protein is not welldefined, but V-minus MV and SeV recombinants have been recovered thatreplicate efficiently in vitro but exhibit reduced replication in vivo(Delenda, et al., Virology 228:55-62, 1997; Delenda et al., Virology242:327-337, 1998; Kato et al., 1997a, supra; Kato et al., J. Virol.71:7266-7272, 1997b; and Valsamakis et al., J. Virol. 72:7754-7761,1998; each incorporated herein by reference).

The viral genome of PIV also contains extragenic leader and trailerregions, possessing all or part of the promoters required for viralreplication and transcription, as well as non-coding and intergenicregions. Thus, the PIV genetic map is represented as 3′leader-N-P/C/D/V-M-F-HN-L-5′ trailer. Some viruses, such as simian virus5 and mumps virus, have a gene located between F and HN that encodes asmall hydrophobic (SH) protein of unknown function. Transcriptioninitiates at the 3′ end and proceeds by a sequential stop-startmechanism that is guided by short conserved motifs found at the geneboundaries. The upstream end of each gene contains a gene-start (GS)signal, which directs initiation of its respective mRNA. The downstreamterminus of each gene contains a gene-end (GE) motif which directspolyadenylation and termination. Exemplary sequences have been describedfor the human PIV3 strains JS (GenBank accession number Z11575,incorporated herein by reference) and Washington (Galinski M. S., in TheParamyxoviruses, Kingsbury, D. W., ed., pp. 537-568, Plenum Press, NewYork, 1991, incorporated herein by reference), and for the bovine PIV3strain 910N (GenBank accession number D80487, incorporated herein byreference).

As used herein, “PIV gene” generally refers to a portion of the PIVgenome encoding an mRNA and typically begins at the upstream end with agene-start (GS) signal and ends at the downstream end with the gene-end(GE) signal. The term PIV gene also includes what is described as“translational open reading frame”, or ORF, particularly in the casewhere a protein, such as C, is expressed from an additional ORF ratherthan from a unique mRNA. To construct human-bovine chimeric PIV of theinvention, one or more PIV gene(s) or genome segment(s) may be deleted,inserted or substituted in whole or in part. This means that partial orcomplete deletions, insertions and substitutions may include openreading frames and/or cis-acting regulatory sequences of any one or moreof the PIV genes or genome segments. By “genome segment” is meant anylength of continuous nucleotides from the PIV genome, which might bepart of an ORF, a gene, or an extragenic region, or a combinationthereof.

The instant invention involves a method for developing live attenuatedPIV vaccine candidates based on chimeras between HPIVs and BPIV3.Chimeras are constructed through a cDNA-based virus recovery system.Recombinant viruses made from cDNA replicate independently and arepropagated in the same manner as if they were biologically-derivedviruses. Preferred human-bovine chimeric PIV vaccine candidates of theinvention bear one or more of the major antigenic determinants of one ormore human PIV(s), e.g., HPIV1, HPIV2, and/or HPIV3, in a backgroundwhich is attenuated by the substitution or addition of one or more BPIVgenes or genome segments. The major protective antigens of PIVs aretheir HN and F glycoproteins, although other proteins can alsocontribute to a protective immune response.

Thus, the invention provides a new basis for attenuating a wild type ormutant parental virus for use as a vaccine against PIV, one which isbased on host range effects due to the introduction of one or moregene(s) or genome segment(s) between HPIV and BPIV. There are numerousnucleotide and amino acid sequence differences between BPIV and HPIV,which are reflected in host range differences. For example, betweenHPIV3 and BPIV3 the percent amino acid identity for each of thefollowing proteins is: N (86%), P (65%), M (93%), F (83%), HN (77%), andL (91%). The host range difference is exemplified by the highlypermissive growth of HPIV3 in rhesus monkeys, compared to the restrictedreplication of two different strains of BPIV3 in the same animal (vanWyke Coelingh et al., 1988, supra). Although the basis of the host rangedifferences between HPIV3 and BPIV3 remains to be deterrnined, it islikely that they will involve more than one gene and multiple amino aciddifferences. The involvement of multiple genes and possibly cis-actingregulatory sequences, each involving multiple amino acid or nucleotidedifferences, gives a very broad basis for attenuation, one which cannotreadily be altered by reversion. This is in contrast to the situationwith other live attenuated HPIV3 viruses which are attenuated by one orseveral point mutations. In this case, reversion of any individualmutation may yield a significant reacquisition of virulence or, in acase where only a single residue specified attenuation, completereacquisition of virulence.

In exemplary embodiments of the invention described hereinbelow, thebackground genome or antigenome is an HPIV3 genome or antigenome, andthe heterologous gene or genome segment is a N ORF derived from,alternatively, a Ka or SF strain of BPIV3 (which are 99% related inamino acid sequence). The N ORF of the HPIV3 background antigenome issubstituted by the counterpart BPIV3 N ORF yielding a novel recombinanthuman-bovine chimeric PIV cDNA clone. Replacement of the HPIV3 N ORF ofHPIV3 with that of BPIV3 Ka or SF results in a protein withapproximately 70 amino acid differences (depending on the straininvolved) from that of HPIV3 N. N is one of the more conserved proteins,and substitution of other proteins such as P, singly or in combination,would result in many more amino acid differences. The involvement ofmultiple genes and genome segments each conferring multiple amino acidor nucleotide differences provides a broad basis for attenuation whichis highly stable to reversion.

This mode of attenuation contrasts sharply to current HPIV vaccinecandidates that are attenuated by one or more point mutations, wherereversion of an individual mutation may yield a significant or completereacquisition of virulence. In addition, several known attenuating pointmutations in HPIV typically yield a temperature sensitive phenotype. Oneproblem with attenuation associated with temperature sensitivity is thatthe virus can be overly restricted for replication in the lowerrespiratory tract while being under attenuated in the upper respiratorytract. This is because there is a temperature gradient within therespiratory tract, with temperature being higher (and more restrictive)in the lower respiratory tract and lower (less restrictive) in the upperrespiratory tract. The ability of an attenuated virus to replicate inthe upper respiratory tract can result in complications includingcongestion, rhinitis, fever and otitis media, whereas overattenuation inthe lower respiratory tract can reduce immunogenicity. Thus, attenuationachieved solely by temperature sensitive mutations may not be ideal. Incontrast, host range mutations present in human-bovine chimeric PIV ofthe invention will not in most cases confer temperature sensitivity.Therefore, the novel method of PIV attenuation provided by the inventionwill be more stable genetically and phenotypically and less likely to beassociated with residual virulence in the upper respiratory tractcompared to other known PIV vaccine candidates.

Surprisingly, both the Ka and SF HPIV3/BPIV3 chimeric recombinantsinvolving the N ORF replacement were viable. Since the N protein of Kaor SF strain BPIV3 differs in 70 of 515 amino acid residues,respectively, from that of the JS strain of HPIV3. It was thereforeunexpected that a bovine N protein with this level of amino acidsequence divergence could efficiently interact with the HPIV3 RNA, orwith other HPIV3 proteins that constitute the functionalreplicase/transcriptase. Equally surprising was the finding that the Kaand SF chimeric viruses replicated as efficiently in cell culture aseither HPIV3 or BPIV3 parent indicating that the chimeric recombinantsdid not exhibit gene incompatibilities that restricted replication invitro. This property of efficient replication in vitro is importantsince it permits efficient manufacture of this biological.

Also surprising is the observation, based on the studies hereinbelow,that the Ka and the SF HPIV3/BPIV3 chimeric recombinants (termed cKa andcSF), bearing only one bovine gene, are nearly equivalent to their BPIV3parents in the degree of host range restriction in the respiratory tractof the rhesus monkey. In particular, the cKa and cSF viruses exhibitedapproximately a 60-fold or 30-fold reduction, respectively, inreplication in the upper respiratory tract of rhesus monkeys compared toreplication of HPIV3. Based on this finding, it is possible that otherBPIV3 genes will also confer desired levels of host range restrictionwithin human-bovine chimeric PIV of the invention. Thus, according tothe methods herein, a list of attenuating determinants will be readilyidentified in heterologous genes and genome segments of both HPIV andBPIV that will confer, in appropriate combination, an optimal level ofhost range restriction and immunogenicity on human-bovine chimeric PIVselected for vaccine use. In preferred vaccine recombinants, attenuationmarked by replication in the lower and/or upper respiratory tract in anaccepted animal model for PIV replication in humans, e.g., hamsters orrhesus monkeys, may be reduced by at least about two-fold, more oftenabout 5-fold, 10-fold, or 20-fold, and preferably 50-100-fold and up to1,000-fold or greater overall (e.g., as measured between 3-8 daysfollowing infection) compared to growth of the corresponding wild-typeor mutant parental PIV strain.

Confirming the unexpected nature and advantages provided by thehuman-bovine chimeric PIV of the invention, both the cKa and cSF induceda high level of protection against HPIV3 challenge in the respiratorytract of rhesus monkeys, despite the exceptional degree of restrictionof replication exhibited by these viruses in this model for human PIVinfection and protection. In particular, previous infection with eitherchimeric virus induced a high level of resistance to replication of therJS challenge virus in both the upper and lower respiratory tract.Infection of monkeys with cKa elicited a high degree of protection asindicated by an approximate 300-fold reduction of replication of wildtype HPIV3 (rJS) in the upper respiratory tract, and an approximate1000-fold reduction in the lower tract compared to uninoculated controlmonkeys. Monkeys infected with cSF manifested a 2000-fold reduction ofreplication of rJS in the upper respiratory tract, and a 1000-foldreduction in the lower tract compared to uninoculated control monkeys.The levels of protection elicited by cKa or cSF were comparable to thoseseen in monkeys previously infected with either the bovine or the humanPIV parent. Thus, infection with human-bovine chimeric PIV of theinvention provides a high level of protection in the upper and lowerrespiratory tract of monkeys, and both chimeric viruses representpromising vaccine candidates. In other preferred vaccine recombinants,the immunogenic activity of human-bovine chimeric PIV will be balancedagainst the level of attenuation to achieve useful vaccine candidates,and will typically be marked by a reduction of replication of challengevirus, e.g., rJS in the lower and/or upper respiratory tract by about50-100-fold, 100-500-fold, preferably about 500-2,000-fold and up to3,000-fold or greater overall (e.g., as measured between 3-8 dayspost-challenge). Thus, the recombinant vaccine viruses of the inventionmaintain immunogenicity while exhibiting concomitant reductions inreplication and growth. This surprising assemblage of phenotypic traitsis highly desired for vaccine development.

The observation that the N gene from two independent strains of BPIV3confers an attenuation phenotype on HPIV3 for the rhesus monkeyindicates that this is likely a property shared by N genes of other BPIVstrains. Accordingly, within the methods of the invention any BPIV geneor genome segment, singly or in combination with one or more other BPIVgene(s) or genome segment(s), can be combined with HPIV sequences toproduce an attenuated HPIV3/BPIV3 chimeric recombinant virus suitablefor use as a vaccine virus. In preferred embodiments, all HPIVs,including HPIV1, HPIV2, HPIV3. and variant strains thereof, are usefulrecipients for attenuating BPIV gene(s) and/or genome segment(s). Ingeneral, the HPIV genes selected for inclusion in a HPIV3/BPIV3 chimericvirus will include one or more of the protective antigens, such as theHN or F glycoproteins.

Alternative human-bovine chimeric PIV of the invention will containprotective antigenic determinants of HPIV1 or HPIV2. This may beachieved, for example, by expression of an HN and/or F gene of HPIV1 orHPIV2 as an extra gene(s) in an attenuated HPIV3/BPIV3 chimericrecombinant. Alternatively, it is possible to use a HPIV3/HPIV1 or aHPIV3/HPIV2 antigenic chimeric virus, in which the HPIV1 or HPIV2 HNand/or F genes replace their PIV3 counterpart(s) (Skiadopoulos et al.,1999a, supra; Tao et al., 1999, supra; and U.S. patent application Ser.No. 09/083,793, filed May 22, 1998; each incorporated herein byreference), as a recipient or background virus for one or moreheterologous, attenuating bovine gene(s) or genome segment(s), forexample a Ka or SF N gene or genome segment. Such antigenic chimericviruses will be attenuated by the bovine N gene, but will induceimmunity to the HPIV1 or HPIV2 virus. In this context, a chimeric PIV1vaccine candidate has been generated using the PIV3 cDNA rescue systemby replacing the PIV3 HN and F open reading frames (ORFs) with those ofPIV1 in a PIV3 full-length cDNA that contains the three attenuatingmutations in L. The recombinant chimeric virus derived from this cDNA isdesignated rPIV3-1.cp45L (Skiadopoulos et al., 1998, supra; Tao et al.,1998, supra; Tao et al., 1999, supra). rPIV3-1.cp45L was attenuated inhamsters and induced a high level of resistance to challenge with PIV1.A recombinant chimeric virus, designated rPIV3-1cp45, has also beenproduced that contains 12 of the 15 cp45 mutations, i.e., excluding themutations in HN and F, and is highly attenuated in the upper and lowerrespiratory tract of hamsters (Skiadopoulos et al., 1999a, supra).

Still other HPIV/BPIV chimeric recombinants will incorporate two or moreBPIV genes or genome segments, in any combination, up to and includingall of the BPIV genome other than selected genes or antigenicdeterminants selected from HN or F gene(s) and genome segment(s), whichcould come from a human HPIV1, HPIV2, or HPIV3 virus. Yet additionalembodiments of the invention are directed to human-bovine chimeric PIVincorporating attenuating genes from other animal PIVs, such as murinePIV1, the canine SV5 PIV2 virus, or another avian or mammalian PIV incombination with a HPIV backbone, alternatively including a chimericHPIV backbone, from HPIV1, HPIV2, and/or HPIV3.

In other detailed aspects of the invention, human-bovine chimeric PIVare employed as vectors for protective antigens of heterologouspathogens, including other PIVs and non-PIV viruses and non-viralpathogens. Within these aspects, the bovine-human chimeric genome orantigenome comprises a partial or complete PIV “vector genome orantigenome” combined with one or more heterologous genes or genomesegments encoding one or more antigenic determinants of one or moreheterologous pathogens (see, e.g., U.S. Provisional Patent ApplicationSer. No. 60/170,195, filed Dec. 10, 1999 by Murphy et al., incorporatedherein by reference). The heterologous pathogen in this context may be aheterologous PIV and the heterologous gene(s) or genome segment(s) canbe selected to encodes one or more PIV N, P, C, D, V, M, F, SH (whereapplicable), HN and/or L protein(s), as well as protein domains,fragments, and immunogenic regions or epitopes. PIV vector vaccines thusconstructed may elicit a polyspecific immune response and may beadministered simultaneously or in a coordinate adminstration protocolwith other vaccine agents.

In exemplary embodiments of the invention, human-bovine chimeric PIV maycomprise a vector genome or antigenome that is a partial or completeHPIV genome or antigenome, which is combined with or is modified toincorporate one or more heterologous genes or genome segments encodingantigenic determinant(s) of one or more heterologous PIV(s), includingheterologous HPIVs selected from HPIV1, HPIV2, or HPIV3. In moredetailed aspects, the vector genome or antigenome is a partial orcomplete HPIV3 genome or antigenome and the heterologous gene(s) orgenome segment(s) encoding the antigenic determinant(s) is/are of one ormore heterologous HPIV(s). Typically, the chimeric genome or antigenomeincorporates one or more gene(s) or genome segment(s) of a BPIV thatspecifies attenuation.

In exemplary aspects of the invention, the bovine-human chimeric PIVincorporates one or more HPIV1 or HPIV2 genes or genome segments thatencode(s) one or more HN and/or F glycoproteins or antigenic domains,fragments or epitopes thereof within a partial or complete HPIV3 vectorgenome or antigenome. In more detailed aspects, both HPIV1 genesencoding HN and F glycoproteins are substituted for counterpart HPIV3 HNand F genes to form a chimeric HPIV3-1 vector genome or antigenome. Suchrecombinant constructs can be used to produce vaccine virus directly, orcan be further modified by addition or incorporation of one or moregenes or gene segments encoding one or more antigenic determinants. Suchconstructs for the production of vaccine viruses typically incorporateone or more heterologous gene(s) or genome segment(s) of a BPIV thatspecifies attenuation, for example an open reading frame (ORF) encodingan attenuating BPIV protein, such as N. Certain human-bovine chimericPIV of the invention may be employed as vectors for generating specificvaccines to HPIV2, for example wherein a transcription unit comprisingan open reading frame (ORF) of an HPIV2 HN gene is added to orincorporated within a chimeric HPIV3-1 vector genome or antigenome andthe chimeric construct is attenuated by incorporation of a BPIV gene orgenome segment.

Within related aspects of the invention, the vector genome or antigenomeis a partial or complete BPIV genome or antigenome, and the heterologousgenes or genome segments encoding the antigenic determinant(s) is/are ofone or more HPIV(s). Typically, the determinant(s) is/are selected fromHPIV1, HPIV2 or HPIV3 HN and F glycoproteins, but antigenic domains,fragments and epitopes of these and other antigenic proteins are alsouseful. In certain embodiments, one or more genes or genome segmentsencoding one or more antigenic determinant(s) of HPIV2 is/are added toor substituted within the partial or complete BPIV vector genome orantigenome. Alternatively, a plurality of heterologous genes or genomesegments encoding antigenic determinants of multiple HPIVs may be addedto or incorporated within the partial or complete BPIV vector genome orantigenome.

In yet additional aspects of the invention, human-bovine chimeric PIVare provided as vectors for a range of non-PIV pathogens (see, e.g.,U.S. Provisional Patent Application Ser. No. 60/170,195, filed Dec. 10,1999 by Murphy et al., incorporated herein by reference). The vectorgenome or antigenome for use within these aspects of the invention maycomprise a partial or complete BPIV or HPIV genome or antigenome, andthe heterologous pathogen may be selected from measles virus, subgroup Aand subgroup B respiratory syncytial viruses, mumps virus, humanpapilloma viruses, type 1 and type 2 human immunodeficiency viruses,herpes simplex viruses, cytomegalovirus, rabies virus, Epstein Barrvirus, filoviruses, bunyaviruses, flaviviruses, alphaviruses andinfluenza viruses.

For example, a HPIV or BPIV vector genome or antigenome for constructingbovine-human chimeric PIV of the invention may incorporate heterologousantigenic determinant(s) selected from the measles virus HA and Fproteins, or antigenic domains, fragments and epitopes thereof. Inexemplary embodiments, a transcription unit comprising an open readingframe (ORF) of a measles virus HA gene is added to or incorporatedwithin a BPIV or HPIV3 vector genome or antigenome.

Alternatively bovine-human chimeric PIV of the invention may used asvectors to incorporate heterologous antigenic determinant(s) fromrespiratory syncytial virus (RSV), for example by incorporating one ormore genes or genome segments that encode(s) RSV F and/or G glycoproteinor immunogenic domain(s) or epitope(s) thereof. In this context, thecloning of RSV cDNA and other disclosure is provided in U.S. ProvisionalPatent Application No. 60/007,083, filed Sep. 27, 1995; U.S. patentapplication Ser. No. 08/720,132, filed Sep. 27, 1996; U.S. ProvisionalPatent Application No. 60/021,773, filed Jul. 15, 1996; U.S. ProvisionalPatent Application No. 60/046,141, filed May 9, 1997; U.S. ProvisionalPatent Application No. 60/047,634, filed May 23, 1997; U.S. patentapplication Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding toInternational Publication No. WO 98/02530); U.S. patent application Ser.No. 09/291,894, filed on Apr. 13, 1999; U.S. Provisional PatentApplication Ser. No. 60/129,006, filed on Apr. 13, 1999; Collins, etal., 1995, supra; Bukreyev, et al., J. Virol. 70:6634-6641, 1996; Juhaszet al., 1997, supra; Durbin et al., 1997a, supra; He et al., 1997,supra; Baron et al., 1997, supra; Whitehead et al., 1998a, supra;Whitehead et al., 1998b, supra; Jin et al., 1998, supra; and Whiteheadet al., 1999, supra; and Bukreyev et al., Proc. Natl. Acad. Sci. USA96:2367-2372, 1999, each incorporated herein by reference in itsentirety for all purposes).

According to this aspect of the invention, human-bovine chimeric PIV areprovided which incorporate at least one antigenic determinant from aheterologous PIV or non-PIV pathogen. For example, one or moreindividual gene(s) or genome segment(s) of HPIV3 may be replaced withcounterpart gene(s) or genome segment(s) from human RSV, or an RSV geneor genome segment can be inserted or added as an supernumerary gene.Alternatively, a selected, heterologous genome segment, e.g. encoding acytoplasmic tail, transmembrane domain or ectodomain of an RSVglycoprotein, is substituted for a counterpart genome segment in, e.g.,the same gene in HPIV3 or within a different gene in HPIV3, or addedwithin a non-coding sequence of the HPIV3 genome or antigenome to yielda chimeric PIV-RSV glycoprotein. In one embodiment, a genome segmentfrom an F gene of human RSV is substituted for a counterpart HPIV3genome segment to yield constructs encoding chimeric proteins, e.g.fusion proteins having a cytoplasmic tail and/or transmembrane domain ofPIV fused to an ectodomain of RSV to yield a novel attenuated virus,and/or a multivalent vaccine immunogenic against both PIV and RSV.

As noted above, it is often desirable to adjust the attenuationphenotype in human-bovine chimeric PIV of the invention by introducingadditional mutations that increase or decrease attenuation or otherwisealter the phenotype of the chimeric virus. Detailed descriptions of thematerials and methods for producing recombinant PIV from cDNA, and formaking and testing the full range of mutations and nucleotidemodifications set forth herein as supplemental aspects of the presentinvention, provided in, e.g., Durbin et al., 1997a, supra; U.S. patentapplication Ser. No. 09/083,793, filed May 22, 1998; U.S. ProvisionalApplication No. 60/047,575, filed May 23, 1997 (corresponding toInternational Publication No. WO 98/53078), and U.S. ProvisionalApplication No. 60/059,385, filed Sep. 19, 1997; each incorporatedherein by reference. In particular, these documents describe methods andprocedures for mutagenizing, isolating and characterizing PIV to obtainattenuated mutant strains (e.g., temperature sensitive (ts), coldpassaged (cp) cold-adapted (ca), small plaque (sp) and host-rangerestricted (hr) mutant strains) and for identifying the genetic changesthat specify the attenuated phenotype. In conjunction with thesemethods, the foregoing documents detail procedures for determiningreplication, immunogenicity, genetic stability and protective efficacyof biologically derived and recombinantly produced attenuated human PIVin accepted model systems, including murine and non-human primate modelsystems. In addition, these documents describe general methods fordeveloping and testing immunogenic compositions, including monovalentand bivalent vaccines, for prophylaxis and treatrnent of PIV infection.Methods for producing infectious recombinant PIV by construction andexpression of cDNA encoding a PIV genome or antigenome coexpressed withessential PIV proteins are also described in the above-incorporateddocuments, which include description of the following exemplary plasmidsthat may be employed to produce infectious PIV viral clones: p3/7(131)(ATCC 97990); p3/7(131)2G (ATCC 97889); and p218(131) (ATCC 97991); eachdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers.

Also disclosed in the above-incorporated references are methods forconstructing and evaluating infectious recombinant PIV that are modifiedto incorporate phenotype-specific mutations identified inbiologically-derived PIV mutants, e.g., cold passaged (cp), cold adapted(ca), host range restricted (hr), small plaque (sp), and/or temperaturesensitive (ts) mutants, for example the JS HPIV3 cp 45 mutant strain.Other mutations may be attenuating without an auxiliary markerphenotype. Mutations identified in these mutants can be readily adoptedin human-bovine chimeric PIV. In exemplary embodiments, one or moreattenuating mutations occur in the polymerase L protein, e.g., at aposition corresponding to Tyr₉₄₂, Leu₉₉₂, or Thr₁₅₅₈ of JS cp45.Preferably, these mutations are incorporated in human-bovine chimericPIV of the invention by an identical, or conservative, amino acidsubstitution as identified in the biological mutant. Thus, PIVrecombinants may incorporate a mutation wherein Tyr₉₄₂ is replaced byHis, Leu₉₉₂ is replaced by Phe, and/or Thr1558 is replaced by Ile.Substitutions that are conservative to these replacement amino acids arealso useful to achieve a desired mutant phenotype

Other exemplary mutations adopted from a biologically derived PIV mutantinclude one or more mutations in the N protein, including specificmutations at a position corresponding to residues Val₉₆ or Ser₃₈₉ of JScp45. In more detailed aspects, these mutations are represented as Val₉₆to Ala or Ser₃₈₉ to Ala or substitutions that are conservative thereto.Also useful within recombinant PIV of the invention are amino acidsubstitution in the C protein, e.g., a mutation at a positioncorresponding to Ile₉₆ of JS cp45, preferably represented by anidentical or conservative substitution of Ile₉₆ to Thr. Furtherexemplary mutations adopted from biologically derived PIV mutantsinclude one mutation in the M gene such as Pro₁₉₉ in JS cp45, or moremutations in the F protein, including mutations adopted from JS cp45 ata position corresponding to residues Ile420 or Ala₄₅₀ so of JS cp45,preferably represented by acid substitutions Ile₄₂₀ to Val or Ala₄₅₀ toThr or substitutions conservative thereto. Other human-bovine chimericPIV within the invention adopt one or more amino acid substitutions inthe HN protein, as exemplified hereinbelow by a recombinant PIV adoptinga mutation at a position corresponding to residue Val₃₈₄ of JS,preferably represented by the substitution Val₃₈₄ to Ala.

Yet additional examples within this aspect of the invention includehuman-bovine chimeric PIV which incorporate one or more mutations innoncoding portions of the PIV genome or antigenome, for example in a 3′leader sequence. Exemplary mutations in this context may be engineeredat a position in the 3′ leader of a recombinant virus at a positioncorresponding to nucleotide 23, 24, 28, or 45 of JS cp45. Yet additionalexemplary mutations may be engineered in the N gene start sequence, forexample by changing one or more nucleotides in the N gene startsequence, e.g., at a position corresponding to nucleotide 62 of JS cp45.In more detailed aspects human-bovine chimeric PIV incorporate a T to Cchange at nucleotide 23, a C to T change at nucleotide 24, a G to Tchange at nucleotide 28, and/or a T to A change at nucleotide 45.Additional mutations in extragenic sequences are exemplified by a A to Tchange in N gene start sequence at a position corresponding tonucleotide 62 of JS.

These foregoing exemplary mutations which can be engineered in ahuman-bovine chimeric PIV of the invention have been successfullyengineered and recovered in recombinant PIV, as represented by therecombinant PIV clones designated rcp45, rcp45 L, rcp45 F, rcp45 M,rcp45 HN, rcp45 C, rcp45 F, rcp45 3′N, 3′NL, and rcp45 3′NCMFHN (Durbinet al., 1997a, supra; Skiadopolos et al., 1998, supra; Skiadopolos etal., J. Virol. 73:1374-1381, 1999b; U.S. patent application Ser. No.09/083,793, filed May 22, 1998; U.S. Provisional Application No.60/047,575, filed May 23, 1997 (corresponding to InternationalPublication No. WO 98/53078), and U.S. Provisional Application No.60/059,385, filed Sep. 19, 1997, each incorporated herein by reference).In addition, the above-incorporated references describe construction ofchimeric PIV recombinants, e.g., having the HN and F genes of HPIV1substituted into a partial HPIV3 background genome or antigenome, whichis further modified to bear one or more of the attenuating mutationsidentified in HPIV3 JS cp45. One such chimeric recombinant incorporatesall of the attenuating mutations identified in the L gene of cp45. Ithas since been shown that all of the cp45 mutations outside of theheterologous (HPIV1) HN and F genes can be incorporated in a HPIV3-lrecombinant to yield an attenuated, chimeric vaccine candidate.

From JS cp45 and other biologically derived PIV mutants, a large “menu”of attenuating mutations is provided, each of which can be combined withany other mutation(s) for adjusting the level of attenuation,immunogenicity and genetic stability in a recombinant PIV bearing C, D,and/or V deletion or knock out mutation(s). In this context, manyrecombinant PIVs of the invention will include one or more, andpreferably two or more, mutations from biologically derived PIV mutants,e.g., any one or combination of mutations identified in JS cp45.Preferred PIV recombinants within the invention will incorporate aplurality and up to a full complement of the mutations present in JScp45 or other biologically derived mutant PIV strains. Preferably, thesemutations are stabilized against reversion in human-bovine, chimeric PIVby multiple nucleotide substitutions in a codon specifying eachmutation.

Additional mutations that may be incorporated in human-bovine chimericPIV of the invention are mutations, e.g., attenuating mutations,identified in heterologous PIV or more distantly related nonsegmentednegative stranded RNA viruses. In particular, attenuating and otherdesired mutations identified in one negative stranded RNA virus may be“transferred”, e.g., introduced by mutagenesis in a correspondingposition within the genome or antigenome of the human-bovine chimericPIV. Briefly, desired mutations in one heterologous negative strandedRNA virus are transferred to the PIV recipient (e.g., bovine or humanPIV, respectively). This involves mapping the mutation in theheterologous virus, thus identifying by sequence alignment thecorresponding site in the recipient RSV, and mutating the nativesequence in the PIV recipient to the mutant genotype (either by anidentical or conservative mutation), as described in PCT/US00/09695filed Apr. 12, 2000 and its priority U.S. Provisional Patent ApplicationSer. No. 60/129,006, filed Apr. 13, 1999, incorporated herein byreference). As this disclosure teaches, it is preferable to modify therecipient genome or antigenome to encode an alteration at the subjectsite of mutation that corresponds conservatively to the alterationidentified in the heterologous mutant virus. For example, if an aminoacid substitution marks a site of mutation in the mutant virus comparedto the corresponding wild-type sequence, then a similar substitutionshould be engineered at the corresponding residue(s) in the recombinantvirus. Preferably the substitution will involve an identical orconservative amino acid to the substitute residue present in the mutantviral protein. However, it is also possible to alter the native aminoacid residue at the site of mutation non-conservatively with respect tothe substitute residue in the mutant protein (e.g., by using any otheramino acid to disrupt or impair the function of the wild-type residue).

Negative stranded RNA viruses from which exemplary mutations areidentified and transferred into human-bovine chimeric PIV of theinvention include other PIVs (e.g., HPIV1, HPIV2, HPIV3, HPIV4A, HPIV4Band BPIV3), RSV, Sendai virus (SeV), Newcastle disease virus (NDV),simian virus 5 (SV5), measles virus (MeV), rinderpest virus, caninedistemper virus (CDV), rabies virus (RaV) and vesicular stomatitis virus(VSV), among others.

A variety of exemplary mutations for use within the invention aredisclosed in the above-incorporated reference, including but not limitedto an amino acid substitution of phenylalanine at position 521 of theRSV L protein corresponding to and therefore transferable to asubstitution of phenylalanine (or a conservatively related amino acid)at position 456 of the HPIV3 L protein. In the case of mutations markedby deletions or insertions, these can be introduced as correspondingdeletions or insertions into the recombinant virus, either within thebackground genome or antigenome or within the heterologous gene orgenome segment incorporated therein. However the particular size andamino acid sequence of the deleted or inserted protein fragment canvary.

Yet additional human-bovine PIV vaccine candidates within the inventioncan be achieved by modifying the chimeric PIV genome or antigenome toencode an analogous mutation to an attenuating mutation identified inSendai virus (SeV). In one example, the attenuating mutation comprisesan amino acid substitution of phenylalanine at position 170 of the Cprotein of SeV. The PIV genome or antigenome is modified to encode analteration of a conserved residue that corresponds conservatively to thealteration marking the attenuating mutation in the heterologous, SeVmutant. In one embodiment, the mutation is incorporated within arecombinant HPIV3 protein and comprises an amino acid substitution ofphenylalanine at position 164 of the C protein of HPIV3.

Various target proteins are amenable to introduction of attenuatingmutations from one negative stranded RNA virus at a corresponding sitewithin chimeric human-bovine PIV of the invention. Throughout the orderMononegavirales, five target proteins are strictly conserved and showmoderate to high degrees of sequence identity for specific regions ordomains. In particular, all known members of the order share ahomologous constellation of five proteins: a nucleocapsid protein (N), anucleocapsid phosphoprotein (P), a nonglycosylated matrix (M) protein,at least one surface glycoprotein (HN, F, H, or G) and a largepolymerase (L) protein. These proteins all represent useful targets forincorporating attenuating mutations by altering one or more conservedresidues in a protein of the recombinant virus at a site correspondingto the site of an attenuating mutation identified in the heterologous,mutant virus.

In this context, the methods for transferring heterologous mutationsinto chimeric human-bovine PIV of the invention are based onidentification of an attenuating mutation in a first negative strandedRNA virus. The mutation, identified in terms of mutant versus wild-typesequence at the subject amino acid position(s) marking the site of themutation, provides an index for sequence comparison against a homologousprotein in the chimeric virus (either in the background genome orantigenome or in the heterologous gene or gene segment added orsubstituted therein) that is the target for recombinant attenuation. Theattenuating mutation may be previously known or may be identified bymutagenic and reverse genetics techniques applied to generate andcharacterize biologically-derived mutant virus. Alternatively,attenuating mutations of interest may be generated and characterized denovo, e.g., by site directed mutagenesis and conventional screeningmethods.

Each attenuating mutation identified in a-negative stranded RNA virusprovides an index for sequence comparison against a homologous proteinin one or more heterologous negative stranded virus(es). In thiscontext, existing sequence alignments may be analyzed, or conventionalsequence alignment methods may be employed to yield sequence comparisonsfor analysis, to identify corresponding protein regions and amino acidpositions between the protein bearing the attenuating mutation and ahomologous protein of a different virus that is the target recombinantvirus for attenuation. Where one or more residues marking theattenuating mutation have been altered from a “wild-type” identity thatis conserved at the corresponding amino acid position(s) in the targethuman-bovine chimeric virus protein, the genome or antigenome of thetarget virus is recombinantly modified to encode an amino acid deletion,substitution, or insertion to alter the conserved residue(s) in thetarget virus protein and thereby confer an analogous, attenuatedphenotype on the recombinant virus.

Within this rational design method for constructing attenuatedrecombinant negative stranded viruses, the wild-type identity ofresidue(s) at amino acid positions marking an attenuating mutation inone negative stranded RNA virus may be conserved strictly, or byconservative substitution, at the corresponding amino acid position(s)in the target, human-bovine chimeric virus protein. Thus, thecorresponding residue(s) in the target virus protein may be identical,or may be conservatively related in terms of amino acid side-groupstructure and function, to the wild-type residue(s) found to be alteredby the attenuating mutation in the heterologous, mutant virus. In eithercase, analogous attenuation in the recombinant virus may be achievedaccording to the methods of the invention by modifying the recombinantgenome or antigenome of the target virus to encode the amino aciddeletion, substitution, or insertion to alter the conserved residue(s).

In this context, it is preferable to modify the genome or antigenome toencode an alteration of the conserved residue(s) that correspondsconservatively to the alteration marking the attenuating mutation in theheterologous, mutant virus. For example, if an amino acid substitutionmarks a site of mutation in the mutant virus compared to thecorresponding wild-type sequence, then a substitution should beengineered at the corresponding residue(s) in the recombinant virus.Preferably the substitution will be identical or conservative to thesubstitute residue present in the mutant viral protein. However, it isalso possible to alter the native amino acid residue at the site ofmutation non-conservatively with respect to the substitute residue inthe mutant protein (e.g., by using any other amino acid to disrupt orimpair the identity and function of the wild-type residue). In the caseof mutations marked by deletions or insertions, these can transferred ascorresponding deletions or insertions into the recombinant virus,however the particular size and amino acid sequence of the deleted orinserted protein fragment can vary.

Within alternative aspects of the invention, mutations thus transferredfrom heterologous mutant negative standed viruses may confer a varietyof phenotypes within human-bovine chimeric PIV of the invention, inaddition to or associated with the desired, an attenuated phenotype.Thus, exemplary mutations incorporated within recombinant proteins ofthe virus may confer temperature sensitive (ts), cold-adapted (ca),small plaque (sp), or host range restricted (hr) phenotypes, or a changein growth or immunogenicity, in addition to or associated with theattenuated phenotype.

Attenuating mutations in biologically derived PIV and other nonsegmentednegative stranded RNA viruses for incorporation within human-bovinechimeric PIV may occur naturally or may be introduced into wild-type PIVstrains by well known mutagenesis procedures. For example, incompletelyattenuated parental PIV strains can be produced by chemical mutagenesisduring virus growth in cell cultures to which a chemical mutagen hasbeen added, by selection of virus that has been subjected to passage atsuboptimal temperatures in order to introduce growth restrictionmutations, or by selection of a mutagenized virus that produces smallplaques (sp) in cell culture, as described in the above incorporatedreferences.

By “biologically derived PIV” is meant any PIV not produced byrecombinant means. Thus, biologically derived PIV include all naturallyoccurring PIV, including, e.g., naturally occurring PIV having awild-type genomic sequence and PIV having allelic or mutant genomicvariations from a reference wild-type RSV sequence, e.g., PIV having amutation specifying an attenuated phenotype. Likewise, biologicallyderived PIV include PIV mutants derived from a parental PIV by, interalia, artificial mutagenesis and selection procedures.

As noted above, production of a sufficiently attenuated biologicallyderived PIV mutant can be accomplished by several known methods. Onesuch procedure involves subjecting a partially attenuated virus topassage in cell culture at progressively lower, attenuatingtemperatures. For example, partially attenuated mutants are produced bypassage in cell cultures at suboptimal temperatures. Thus, a cp mutantor other partially attenuated PIV strain is adapted to efficient growthat a lower temperature by passage in culture. This selection of mutantPIV during cold-passage substantially reduces any residual virulence inthe derivative strains as compared to the partially attenuated parent.

Alternatively, specific mutations can be introduced into biologicallyderived PIV by subjecting a partially attenuated parent virus tochemical mutagenesis, e.g., to introduce ts mutations or, in the case ofviruses which are already ts, additional ts mutations sufficient toconfer increased attenuation and/or stability of the ts phenotype of theattenuated derivative. Means for the introduction of ts mutations intoPIV include replication of the virus in the presence of a mutagen suchas 5-fluorouridine according to generally known procedures. Otherchemical mutagens can also be used. Attenuation can result from a tsmutation in almost any PIV gene, although a particularly amenable targetfor this purpose has been found to be the polymerase (L) gene.

The level of temperature sensitivity of replication in exemplaryattenuated PIV for use within the invention is determined by comparingits replication at a permissive temperature with that at severalrestrictive temperatures. The lowest temperature at which thereplication of the virus is reduced 100-fold or more in comparison withits replication at the permissive temperature is termed the shutofftemperature. In experimental animals and humans, both the replicationand virulence of PIV correlate with the mutant's shutoff temperature.

The JS cp45 HPIV3 mutant has been found to be relatively stablegenetically, highly immunogenic, and satisfactorily attenuated.Nucleotide sequence analysis of this biologically derived andrecombinant viruses incorporating various individual and combinedmutations found therein, indicates that each level of increasedattenuation is associated with specific nucleotide and amino acidsubstitutions. The above-incorporated references also disclose how toroutinely distinguish between silent incidental mutations and thoseresponsible for phenotype differences by introducing the mutations,separately and in various combinations, into the genome or antigenome ofinfectious PIV clones. This process coupled with evaluation of phenotypecharacteristics of parental aid derivative virus identifies mutationsresponsible for such desired characteristics as attenuation, temperaturesensitivity, cold-adaptation, small plaque size, host range restriction,etc.

Mutations thus identified are compiled into a “menu” and are thenintroduced as desired, singly or in combination, to adjust ahuman-bovine chimeric PIV to an appropriate level of attenuation,immunogenicity, genetic resistance to reversion from an attenuatedphenotype, etc., as desired. In accordance with the foregoingdescription, the ability to produce infectious PIV from cDNA permitsintroduction of specific engineered changes within human-bovine chimericPIV. In particular, infectious, recombinant PIVs are employed foridentification of specific mutation(s) in biologically derived,attenuated PIV strains, for example mutations which specify ts, ca, attand other phenotypes. Desired mutations are thus identified andintroduced into recombinant, human-bovine chimeric PIV vaccine strains.The capability of producing virus from cDNA allows for routineincorporation of these mutations, individually or in various selectedcombinations, into a full-length cDNA clone, whereafter the phenotypesof rescued recombinant viruses containing the introduced mutations canbe readily determined.

By identifying and incorporating specific, biologically derivedmutations associated with desired phenotypes, e.g., a cp or tsphenotype, into infectious PIV clones, the invention provides for other,site-specific modifications at, or within close proximity to, theidentified mutation. Whereas most attenuating mutations produced inbiologically derived PIV are single nucleotide changes, other “sitespecific” mutations can also be incorporated by recombinant techniquesinto biologically derived or recombinant PIV. As used herein,site-specific mutations include insertions, substitutions, deletions orrearrangements of from 1 to 3, up to about 5-15 or more alterednucleotides (e.g., altered from a wild-type PIV sequence, from asequence of a selected mutant PIV strain, or from a parent recombinantPIV clone subjected to mutagenesis). Such site-specific mutations may beincorporated at, or within the region of, a selected, biologicallyderived point mutation. Alternatively, the mutations can be introducedin various other contexts within a PIV clone, for example at or near acis-acting regulatory sequence or nucleotide sequence encoding a proteinactive site, binding site, immunogenic epitope, etc. Site-specific PIVmutants typically retain a desired attenuating phenotype, but mayadditionally exhibit altered phenotypic characteristics unrelated toattenuation, e.g., enhanced or broadened immunogenicity, and/or improvedgrowth. Further examples of desired, site-specific mutants includerecombinant PIV designed to incorporate additional, stabilizingnucleotide mutations in a codon specifying an attenuating pointmutation. Where possible, two or more nucleotide substitutions areintroduced at codons that specify attenuating amino acid changes in aparent mutant or recombinant PIV clone, yielding a biologically derivedor recombinant PIV having genetic resistance to reversion from anattenuated phenotype. In other embodiments, site-specific nucleotidesubstitutions, additions, deletions or rearrangements are introducedupstream or downstream, e.g., from 1 to 3, 5-10 and up to 15 nucleotidesor more 5′ or 3′, relative to a targeted nucleotide position, e.g., toconstruct or ablate an existing cis-acting regulatory element.

In addition to single and multiple point mutations and site-specificmutations, changes to the human-bovine chimeric PIV disclosed hereininclude deletions, insertions, substitutions or rearrangements of one ormore gene(s) or genome segment(s). Particularly useful are deletionsinvolving one or more gene(s) or genome segment(s), which deletions havebeen shown to yield additional desired phenotypic effects for adjustingthe characteristics of human-bovine chimeric PIV within the invention.Thus, U.S. patent application Ser. No. 09/350,821, filed by Durbin etal. on Jul. 9, 1999) describes methods and compositions wherebyexpression of one or more HPIV genes, exemplified by the C, D, and/or VORFs, is reduced or ablated by modifying the PIV genome or anti genometo incorporate a mutation that alters the coding assignment of aninitiation codon or mutation(s) that introduce one or one or more stopcodon(s). Alternatively, one or more of the C, D, and/or V ORFs can bedeleted in whole or in part to render the corresponding protein(s)partially or entirely non-functional or to disrupt protein expressionaltogether. Recombinant PIV having such mutations in C, D, and/or V, orother non-essential gene(s), possess highly desirable phenotypiccharacteristics for vaccine development. For example, thesemodifications may specify one or more desired phenotypic changesincluding (i) altered growth properties in cell culture, (ii)attenuation in the upper and/or lower respiratory tract of mammals,(iii) a change in viral plaque size, (iv) a change in cytopathic effect,and (v) a change in immunogenicity. One such exemplary “knock out”mutant lacking C ORF expression, designated rC-KO, was able to induce aprotective immune response against wild type HPIV3 challenge in anon-human primate model despite its beneficial attenuation phenotype.

Thus, in more detailed aspects of the instant invention, human-bovinechimeric PIV incorporate deletion or knock out mutations in a C, D,and/or V ORF(s) which alters or ablates expression of the selectedgene(s) or genome segment(s). This can be achieved, e.g., by introducinga frame shift mutation or termination codon within a selected codingsequence, altering translational start sites, changing the position of agene or introducing an upstream start codon to alter its rate ofexpression, changing GS and/or GE transcription signals to alterphenotype, or modifying an RNA editing site (e.g., growth, temperaturerestrictions on transcription, etc.). In more detailed aspects of theinvention, human-bovine chimeric PIVs are provided in which expressionof one or more gene(s), e.g., a C, D, and/or V ORF(s), is ablated at thetranslational or transcriptional level without deletion of the gene orof a segment thereof, by, e.g., introducing multiple translationaltermination codons into a translational open reading frame (ORF),altering an initiation codon, or modifying an editing site. These formsof knock-out virus will often exhibit reduced growth rates and smallplaque sizes in tissue culture. Thus, these methods provide yetadditional, novel types of attenuating mutations which ablate expressionof a viral gene that is not one of the major viral protective antigens.In this context, knock-out virus phenotypes produced without deletion ofa gene or genome segment can be alternatively produced by deletionmutagenesis, as described, to effectively preclude correcting mutationsthat may restore synthesis of a target protein. Several other geneknock-outs for the C, D, and/or V ORF(s) deletion and knock out mutantscan be made using alternate designs and methods that are well known inthe art (as described, for example, in (Kretschmer et al., Virology216:309-316, 1996; Radecke et al., Virology 217:418-421, 1996; and Katoet al., 1987a, supra; and Schneider et al., 1997, supra; eachincorporated herein by reference).

These and other nucleotide modifications in human-bovine chimeric PIVmay alter small numbers of bases (e.g., from 15-30 bases, up to 35-50bases or more), large blocks of nucleotides (e.g., 50-100, 100-300,300-500, 500-1,000 bases), or nearly complete or complete genes (e.g.,1,000-1,500 nucleotides, 1,500-2,500 nucleotides, 2,500-5,000,nucleotides, 5,000-6,500 nucleotides or more) in the donor or recipientgenome or antigenome, depending upon the nature of the change (i.e., asmall number of bases may be changed to insert or ablate an immunogenicepitope or change a small genome segment, whereas large block(s) ofbases are involved when genes or large genome segments are added,substituted, deleted or rearranged.

In related aspects, the invention provides for supplementation ofmutations adopted into a recombinant PIV clone from biologically derivedPIV, e.g., cp and ts mutations, with additional types of mutationsinvolving the same or different genes in a further modified PIV clone.Each of the PIV genes can be selectively altered in terms of expressionlevels, or can be added, deleted, substituted or rearranged, in whole orin part, alone or in combination with other desired modifications, toyield a human-bovine chimeric PIV exhibiting novel vaccinecharacteristics. Thus, in addition to or in combination with attenuatingmutations adopted from biologically derived PIV mutants, the presentinvention also provides a range of additional methods for attenuating orotherwise modifying the phenotype of human-bovine chimeric PIV based onrecombinant engineering of infectious PIV clones. A variety ofalterations can be produced in an isolated polynucleotide sequenceencoding a targeted gene or genome segment, including a donor orrecipient gene or genome segment in a chimeric PIV genome or antigenomefor incorporation into infectious clones. More specifically, to achievedesired structural and phenotypic changes in recombinant PIV, theinvention allows for introduction of modifications which delete,substitute, introduce, or rearrange a selected nucleotide or pluralityof nucleotides from a parent genome or antigenome, as well as mutationswhich delete, substitute, introduce or rearrange whole gene(s) or genomesegment(s), within a human-bovine chimeric PIV clone.

Thus provided are modifications in the human-bovine chimeric PIV whichsimply alter or ablate expression of a selected gene, e.g., byintroducing a termination codon within a selected PIV coding sequence oraltering its translational start site or RNA editing site, changing theposition of a PIV gene relative to an operably linked promoter,introducing an upstream start codon to alter rates of expression,modifying (e.g., by changing position, altering an existing sequence, orsubstituting an existing sequence with a heterologous sequence) GSand/or GE transcription signals to alter phenotype (e.g., growth,temperature restrictions on transcription, etc.), and various otherdeletions, substitutions, additions and rearrangements that specifyquantitative or qualitative changes in viral replication, transcriptionof selected gene(s), or translation of selected RNA(s). In this context,any PIV gene or genome segment which is not essential for growth can beablated or otherwise modified in a recombinant PIV to yield desiredeffects on virulence, pathogenesis, immunogenicity and other phenotypiccharacters. As for coding sequences, noncoding, leader, trailer andintergenic regions can be similarly deleted, substituted or modified andtheir phenotypic effects readily analyzed, e.g., by the use ofminireplicons and recombinant PIV.

In addition, a variety of other genetic alterations can be produced in aPIV genome or antigenome for incorporation into human-bovine chimericPIV, alone or together with one or more attenuating mutations adoptedfrom a biologically derived mutant PIV, e.g., to adjust growth,attenuation, immunogenicity, genetic stability or provide otheradvantageous structural and/or phenotypic effects. These additionaltypes of mutations are also disclosed in the foregoing incorporatedreferences and can be readily engineered into human-bovine chimeric PIVof the invention.

In addition to these changes, the order of genes in a human-bovinechimeric PIV can be changed, a PIV genome promoter replaced with itsantigenome counterpart, portions of genes removed or substituted, andeven entire genes deleted. Different or additional modifications in thesequence can be made to facilitate manipulations, such as the insertionof unique restriction sites in various intergenic regions or elsewhere.Nontranslated gene sequences can be removed to increase capacity forinserting foreign sequences.

Other mutations for incorporation into human-bovine chimeric PIV of theinvention include mutations directed toward cis-acting signals, whichcan be identified, e.g., by mutational analysis of PIV minigenomes. Forexample, insertional and deletional analysis of the leader and trailerand flanking sequences identifies viral promoters and transcriptionsignals and provides a series of mutations associated with varyingdegrees of reduction of RNA replication or transcription. Saturationmutagenesis (whereby each position in turn is modified to each of thenucleotide alternatives) of these cis-acting signals also has identifiedmany mutations which affect RNA replication or transcription. Any ofthese mutations can be inserted into a human-bovine chimeric PIVantigenome or genome as described herein. Evaluation and manipulation oftrans-acting proteins and cis-acting RNA sequences using the completeantigenome cDNA is assisted by the use of PIV minigenomes as describedin the above-incorporated references.

Additional mutations within the human-bovine chimeric PIV involvereplacement of the 3′ end of genome with its counterpart fromantigenome, which is associated with changes in RNA replication andtranscription. In one exemplary embodiment, the level of expression ofspecific PIV proteins, such as the protective HN and/or F antigens, canbe increased by substituting the natural sequences with ones which havebeen made synthetically and designed to be consistent with efficienttranslation. In this context, it has been shown that codon usage can bea major factor in the level of translation of mammalian viral proteins(Hans et al., Current Biol. 6:315-324, 1996, incorporated herein byreference). Optimization by recombinant methods of the codon usage ofthe mRNAs encoding the HN and F proteins of PIV, which are the majorprotective antigens, will provide improved expression for these genes.

In another exemplary embodiment, a sequence surrounding a translationalstart site (preferably including a nucleotide in the −3 position) of aselected PIV gene is modified, alone or in combination with introductionof an upstream start codon, to modulate PIV gene expression byspecifying up or down-regulation of translation (Kozak et al., J. Mol.Biol. 196:947-950, 1987). Alternatively, or in combination with otherPIV modifications disclosed herein, gene expression of a human-bovinechimeric PIV can be modulated by altering a transcriptional GS or GEsignal of any selected gene(s) of the virus. In alternative embodiments,levels of gene expression in the human-bovine chimeric PIV are modifiedat the level of transcription. In one aspect, the position of a selectedgene in the PIV gene map can be changed to a more promoter-proximal orpromotor-distal position, whereby the gene will be expressed more orless efficiently, respectively. According to this aspect, modulation ofexpression for specific genes can be achieved yielding reductions orincreases of gene expression from two-fold, more typically four-fold, upto ten-fold or more compared to wild-type levels often attended by acommensurate decrease in expression levels for reciprocally,positionally substituted genes. These and other transpositioning changesyield novel human-bovine chimeric PIV having attenuated phenotypes, forexample due to decreased expression of selected viral proteins involvedin RNA replication, or having other desirable properties such asincreased antigen expression.

Infectious human-bovine chimeric PIV clones of the invention can also beengineered according to the methods and compositions disclosed herein toenhance immunogenicity and induce a level of protection greater thanthat provided by infection with a wild-type PIV or a parent PIV. Forexample, an immunogenic epitope from a heterologous PIV strain or type,or from a non-PIV source such as RSV, can be added to a recombinantclone by appropriate nucleotide changes in the polynucleotide sequenceencoding the genome or antigenome. Alternatively, mutant PIV of theinvention can be engineered to add or ablate (e.g., by amino acidinsertion, substitution or deletion) immunogenic proteins, proteindomains, or forms of specific proteins associated with desirable orundesirable immunological reactions.

Within the methods of the invention, additional genes or genome segmentsmay be inserted into or proximate to the human-bovine chimeric PIVgenome or antigenome. These genes may be under common control withrecipient genes, or may be under the control of an independent set oftranscription signals. Genes of interest include the PIV genesidentified above, as well as non-PIV genes. Non-PIV genes of interestinclude those encoding cytokines (e.g., IL-2 through IL-18, especiallyIL-2, IL-6 and IL-12, IL-18, etc.). gamma-interferon, and proteins richin T helper cell epitopes. These additional proteins can be expressedeither as a separate protein, or as a supernumerary copy of an existingPIV proteins, such as HN or F. This provides the ability to modify andimprove the immune responses against PIV both quantitatively andqualitatively.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or genome segments within a human-bovinechimeric PIV yield highly stable vaccine candidates, which areparticularly important in the case of immunosuppressed individuals. Manyof these changes will result in attenuation of resultant vaccinestrains, whereas others will specify different types of desiredphenotypic changes. For example, accessory (i.e., not essential for invitro growth) genes are excellent candidates to encode proteins thatspecifically interfere with host immunity (see, e.g., Kato et al.,1997a, supra). Ablation of such genes in vaccine viruses is expected toreduce virulence and pathogenesis and/or improve immunogenicity.

In another aspect of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating human-bovine chimericPIV-encoding cDNA) are provided for producing an isolated infectiousPIV. Using these compositions and methods, infectious PIV are generatedfrom a PIV genome or antigenome, a nucleocapsid (N) protein, anucleocapsid phosphoprotein (P), and a large (L) polymerase protein. Inrelated aspects of the invention, compositions and methods are providedfor introducing the aforementioned structural and phenotypic changesinto a recombinant PIV to yield infectious, attenuated vaccine viruses.

Introduction of the foregoing defined mutations into an infectious,human-bovine chimeric PIV clone can be achieved by a variety of wellknown methods. By “infectious clone” with regard to DNA is meant cDNA orits product, synthetic or otherwise, which can be transcribed intogenomic or antigenomic RNA capable of serving as template to produce thegenome of an infectious virus or subviral particle. Thus, definedmutations can be introduced by conventional techniques (e.g.,site-directed mutagenesis) into a cDNA copy of the genome or antigenome.The use of antigenome or genome cDNA subfragments to assemble a completeantigenome or genome cDNA as described herein has the advantage thateach region can be manipulated separately (smaller cDNAs are easier tomanipulate than large ones) and then readily assembled into a completecDNA. Thus, the complete antigenome or genome cDNA, or any subfragmentthereof can be used as template for oligonucleotide-directedmutagenesis. This can be through the intermediate of a single-strandedphagemid form, such as using the Muta-gene® kit of Bio-Rad Laboratories(Richmond, Calif.) or a method using a double-stranded plasmid directlyas template such as the Chameleon mutagenesis kit of Stratagene (LaJolla, Calif.), or by the polymerase chain reaction employing either anoligonucleotide primer or template which contains the mutation(s) ofinterest. A mutated subfragment can then be assembled into the completeantigenome or genome cDNA. A variety of other mutagenesis techniques areknown and available for use in producing the mutations of interest inthe PIV antigenome or genome cDNA. Mutations can vary from singlenucleotide changes to replacement of large cDNA pieces containing one ormore genes or genome regions.

Thus, in one illustrative embodiment mutations are introduced by usingthe Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.In brief, cDNA encoding a portion of a PIV genome or antigenome iscloned into the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome or antigenome. The plasmid containing the genetically alteredgenome or antigenome fragment is then amplified and the mutated piece isthen reintroduced into the full-length genome or antigenome clone.

Infectious PIV of the invention are produced by intracellular orcell-free coexpression of one or more isolated polynucleotide moleculesthat encode a PIV genome or antigenome RNA, together with one or morepolynucleotides encoding viral proteins necessary to generate atranscribing, replicating nucleocapsid. Among the viral proteins usefulfor coexpression to yield infectious PIV are the major nucleocapsidprotein (N) protein, nucleocapsid phosphoprotein (P), large (L)polymerase protein, fusion protein (F), hemagglutinin-neuraminidaseglycoprotein (HN), and matrix (M) protein. Also useful in this contextare products of the C, D and V ORFs of PIV.

cDNAs encoding a PIV genome or antigenome are constructed forintracellular or in vitro coexpression with the necessary viral proteinsto form infectious PIV. By “PIV antigenome” is meant an isolatedpositive-sense polynucleotide molecule which serves as a template forsynthesis of progeny PIV genome. Preferably a cDNA is constructed whichis a positive-sense version of the PIV genome corresponding to thereplicative intermediate RNA, or antigenome, so as to minimize thepossibility of hybridizing with positive-sense transcripts ofcomplementing sequences encoding proteins necessary to generate atranscribing, replicating nucleocapsid.

In some embodiments of the invention the genome or antigenome of arecombinant PIV (rPIV) need only contain those genes or portions thereofnecessary to render the viral or subviral particles encoded therebyinfectious. Further, the genes or portions thereof may be provided bymore than one polynucleotide molecule, i.e., a gene may be provided bycomplementation or the like from a separate nucleotide molecule. Inother embodiments, the PIV genome or antigenome encodes all functionsnecessary for viral growth, replication, and infection without theparticipation of a helper virus or viral function provided by a plasmidor helper cell line.

By “recombinant PIV” is meant a PIV or PIV-like viral or subviralparticle derived directly or indirectly from a recombinant expressionsystem or propagated from virus or subviral particles producedtherefrom. The recombinant expression system will employ a recombinantexpression vector which comprises an operably linked transcriptionalunit comprising an assembly of at least a genetic element or elementshaving a regulatory role in PIV gene expression, for example, apromoter, a structural or coding sequence which is transcribed into PIVRNA, and appropriate transcription initiation and termination sequences.

To produce infectious PIV from a cDNA-expressed PIV genome orantigenome, the genome or antigenome is coexpressed with those PIV N, Pand L proteins necessary to (i) produce a nucleocapsid capable of RNAreplication, and (ii) render progeny nucleocapsids competent for bothRNA replication and transcription. Transcription by the genomenucleocapsid provides the other PIV proteins and initiates a productiveinfection. Alternatively, additional PIV proteins needed for aproductive infection can be supplied by coexpression.

Synthesis of PIV antigenome or genome together with the above-mentionedviral proteins can also be achieved in vitro (cell-free), e.g., using acombined transcription-translation reaction, followed by transfectioninto cells. Alternatively, antigenome or genome RNA can be synthesizedin vitro and transfected into cells expressing PIV proteins.

In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicating PIVnucleocapsid are provided by one or more helper viruses. Such helperviruses can be wild type or mutant. Preferably, the helper virus can bedistinguished phenotypically from the virus encoded by the PIV cDNA. Forexample, it is desirable to provide monoclonal antibodies which reactimmunologically with the helper virus but not the virus encoded by thePIV cDNA. Such antibodies can be neutralizing antibodies. In someembodiments, the antibodies can be used in affinity chromatography toseparate the helper virus from the recombinant virus. To aid theprocurement of such antibodies, mutations can be introduced into the PIVcDNA to provide antigenic diversity from the helper virus, such as inthe HN or F glycoprotein genes.

In alternate embodiments of the invention, the N, P, L and other desiredPIV proteins are encoded by one or more non-viral expression vectors,which can be the same or separate from that which encodes the genome orantigenome. Additional proteins may be included as desired, each encodedby its own vector or by a vector encoding one or more of the N, P, L andother desired PIV proteins, or the complete genome or antigenome.Expression of the genome or antigenome and proteins from transfectedplasmids can be achieved, for example, by each cDNA being under thecontrol of a promoter for T7 RNA polymerase, which in turn is suppliedby infection, transfection or transduction with an expression system forthe T7 RNA polymerase, e.g., a vaccinia virus MVA strain recombinantwhich expresses the T7 RNA polymerase (Wyatt et al., Virology210:202-205, 1995, incorporated herein by reference in its entirety).The viral proteins, and/or T7 RNA polymerase, can also be provided bytransformed mammalian cells or by transfection of preformed mRNA orprotein.

A PIV antigenome may be constructed for use in the present invention by,e.g., assembling cloned cDNA segments, representing in aggregate thecomplete antigenome, by polymerase chain reaction or the like (PCR;described in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCRProtocols. A Guide to Methods and Applications, Innis et al., eds.,Academic Press, San Diego, 1990; each incorporated herein by referencein its entirety) of reverse-transcribed copies of PIV mRNA or genomeRNA. For example, a first construct is generated which comprises cDNAscontaining the left hand end of the antigenome, spanning from anappropriate promoter (e.g., T7 RNA polymerase promoter) and assembled inan appropriate expression vector, such as a plasmid, cosmid, phage, orDNA virus vector. The vector may be modified by mutagenesis and/orinsertion of synthetic polylinker containing unique restriction sitesdesigned to facilitate assembly. For ease of preparation the N, P, L andother desired PIV proteins can be assembled in one or more separatevectors. The right hand end of the antigenome plasmid may containadditional sequences as desired, such as a flanking ribozyme and tandemT7 transcriptional terminators. The ribozyme can be hammerhead type(e.g., Grosfeld et al., J. Virol. 69:5677-5686, 1995), which would yielda 3′ end containing a single nonviral nucleotide, or can be any of theother suitable ribozymes such as that of hepatitis delta virus (Perrottaet al., Nature 350:434-436, 1991), incorporated herein by reference inits entirety) which would yield a 3′ end free of non-PIV nucleotides.The left- and right-hand ends are then joined via a common restrictionsite.

A variety of nucleotide insertions, deletions and rearrangements can bemade in the PIV genome or antigenome during or after construction of thecDNA. For example, specific desired nucleotide sequences can besynthesized and inserted at appropriate regions in the cDNA usingconvenient restriction enzyme sites. Alternatively, such techniques assite-specific mutagenesis, alanine scanning, PCR mutagenesis, or othersuch techniques well known in the art can be used to introduce mutationsinto the cDNA.

Alternative means to construct cDNA encoding the genome or antigenomeinclude reverse transcription-PCR using improved PCR conditions (e.g.,as described in Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699,1994, incorporated herein by reference) to reduce the number of subunitcDNA components to as few as one or two pieces. In other embodimentsdifferent promoters can be used (e.g., T3, SP6) or different ribozymes(e.g., that of hepatitis delta virus. Different DNA vectors (e.g.,cosmids) can be used for propagation to better accommodate the largersize genome or antigenome.

Isolated polynucleotides (e.g., cDNA) encoding the genome or antigenomemay be inserted into appropriate host cells by transfection,electroporation, mechanical insertion, transduction or the like, intocells which are capable of supporting a productive PIV infection, e.g.,HEp-2, FRhL-DBS2, LLC-MK2, MRC-5, and Vero cells. Transfection ofisolated polynucleotide sequences may be introduced into cultured cellsby, for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ed., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., New York, 1987), cationiclipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993)or a commercially available transfection regent, e.g., LipofectACE®(Life Technologies, Gaithersburg, Md.) or the like (each of theforegoing references are incorporated herein by reference in itsentirety).

As noted above, in some embodiments of the invention the N, P, L andother desired PIV proteins are encoded by one or more helper viruseswhich is phenotypically distinguishable from that which encodesthe-genome or antigenome. The N, P, L and other desired PIV proteins canalso be encoded by one or more expression vectors which can be the sameor separate from that which encodes the genome or antigenome, andvarious combinations thereof. Additional proteins may be included asdesired, encoded by its own vector or by a vector encoding one or moreof the N, P, L and other desired PIV proteins, or the complete genome orantigenome.

By providing infectious clones of PIV the invention permits a wide rangeof alterations to be recombinantly produced within the PIV genome (orantigenome), yielding defined mutations which specify desired phenotypicchanges. By “infectious clone” is meant cDNA or its product, syntheticor otherwise, RNA capable of being directly incorporated into infectiousvirions which can be transcribed into genomic or antigenomic RNA capableof serving as a template to produce the genome of infectious viral orsubviral particles. As noted above, defined mutations can be introducedby a variety of conventional techniques (e.g., site-directedmutagenesis) into a cDNA copy of the genome or antigenome. The use ofgenomic or antigenomic cDNA subfragments to assemble a complete genomeor antigenome cDNA as described herein has the advantage that eachregion can be manipulated separately, where small cDNA subjects providefor better ease of manipulation than large cDNA subjects, and thenreadily assembled into a complete cDNA. Thus, the complete antigenome orgenome cDNA, or a selected subfragment thereof, can be used as atemplate for oligonucleotide-directed mutagenesis. This can be throughthe intermediate of a single-stranded phagemid form, such as using theMUTA-gen® kit of Bio-Rad Laboratories (Richmond, Calif.), or a methodusing the double-stranded plasmid directly as a template such as theChameleon® mutagenesis kit of Strategene (La Jolla, Calif.), or by thepolymerase chain reaction employing either an oligonucleotide primer ora template which contains the mutation(s) of interest. A mutatedsubfragment can then be assembled into the complete antigenome or genomecDNA. A variety of other mutagenesis techniques are known and can beroutinely adapted for use in producing the mutations of interest in aPIV antigenome or genome cDNA of the invention.

Thus, in one illustrative embodiment mutations are introduced by usingthe MUTA-gene® phagemid in vitro mutagenesis kit available from Bio-RadLaboratories. In brief, cDNA encoding an PIV genome or antigenome iscloned into the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome or antigenome. The plasmid containing the genetically alteredgenome or antigenome is then amplified.

Mutations can vary from single nucleotide changes to the introduction,deletion or replacement of large cDNA segments containing one or moregenes or genome segments. Genome segments can correspond to structuraland/or functional domains, e.g., cytoplasmic, transmembrane orectodomains of proteins, active sites such as sites that mediate bindingor other biochemical interactions with different proteins, epitopicsites, e.g., sites that stimulate antibody binding and/or humoral orcell mediated immune responses, etc. Useful genome segments in thisregard range from about 15-35 nucleotides in the case of genome segmentsencoding small functional domains of proteins, e.g., epitopic sites, toabout 50, 75, 100, 200-500, and 500-1,500 or more nucleotides.

The ability to introduce defined mutations into infectious PIV has manyapplications, including the manipulation of PIV pathogenic andimmunogenic mechanisms. For example, the functions of PIV proteins,including the N, P, M, F, HN, and L proteins and C, D and V ORFproducts, can be manipulated by introducing mutations which ablate orreduce the level of protein expression, or which yield mutant protein.Various genome RNA structural features, such as promoters, intergenicregions, and transcription signals, can also be routinely manipulatedwithin the methods and compositions of the invention. The effects oftrans-acting proteins and cis-acting RNA sequences can be readilydetermined, for example, using a complete antigenome cDNA in parallelassays employing PIV minigenomes (Dimock et al., J. Virol. 67:2772-2778,1993, incorporated herein by reference in its entirety), whoserescue-dependent status is useful in characterizing those mutants thatmay be too inhibitory to be recovered in replication-independentinfectious virus.

Certain substitutions, insertions, deletions or rearrangements of genesor genome segments within recombinant PIV of the invention (e.g.,substitutions of a genome segment encoding a selected protein or proteinregion, for instance a cytoplasmic tail, transmembrane domain orectodomain, an epitopic site or region, a binding site or region, anactive site or region containing an active site, etc.) are made instructural or functional relation to an existing, “counterpart” gene orgenome segment from the same or different PIV or other source. Suchmodifications yield novel recombinants having desired phenotypic changescompared to wild-type or parental PIV or other viral strains. Forexample, recombinants of this type may express a chimeric protein havinga cytoplasmic tail and/or transmembrane domain of one PIV fused to anectodomain of another PIV. Other exemplary recombinants of this typeexpress duplicate protein regions, such as, duplicate immunogenicregions.

As used herein, “counterpart” genes, genome segments, proteins orprotein regions, are typically from heterologous sources (e.g., fromdifferent PIV genes, or representing the same (i.e., homologous orallelic) gene or genome segment in different PIV types or strains).Typical counterparts selected in this context share gross structuralfeatures, e.g., each counterpart may encode a comparable protein orprotein structural domain, such as a cytoplasmic domain, transmembranedomain, ectodomain, binding site or region, epitopic site or region,etc. Counterpart domains and their encoding genome segments embrace anassemblage of species having a range of size and sequence variationsdefined by a common biological activity among the domain or genomesegment variants.

Counterpart genes and genome segments, as well as other polynucleotidesdisclosed herein for producing recombinant PIV within the invention,often share substantial sequence identity with a selected polynucleotide“reference sequence,” e.g., with another selected counterpart sequence.As used herein, a “reference sequence” is a defined sequence used as abasis for sequence comparison, for example, a segment of a full-lengthcDNA or gene, or a complete cDNA or gene sequence. Generally, areference sequence is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences.

Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith & Waterman (Adv.Appl. Math. 2:482, 1981; incorporated herein by reference), by thehomology alignment algorithm of Needleman & Wunsch, (J. Mol. Biol.48:443, 1970; incorporated herein by reference), by the search forsimilarity method of Pearson & Lipman, (Proc. Natl. Acad. Sci. USA85:2444, 1988; incorporated herein by reference), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., incorporated herein byreference), or by inspection, and the best alignment (i.e., resulting inthe highest percentage of sequence similarity over the comparisonwindow) generated by the various methods is selected. The term “sequenceidentity” means that two polynucleotide sequences are identical (i.e.,on a nucleotide-by-nucleotide basis) over the window of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence.

In addition to these polynucleotide sequence relationships, proteins andprotein regions encoded by recombinant PIV of the invention are alsotypically selected to have conservative relationships, i.e. to havesubstantial sequence identity or sequence similarity, with selectedreference polypeptides. As applied to polypeptides, the term “sequenceidentity” means peptides share identical amino acids at correspondingpositions. The term “sequence similarity” means peptides have identicalor similar amino acids (i.e., conservative substitutions) atcorresponding positions. The term “substantial sequence identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). The term “substantial similarity”means that two peptide sequences share corresponding percentages ofsequence similarity. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Abbreviations for the twenty naturally occurringamino acids used herein follow conventional usage (Immunology—ASynthesis, 2nd ed., E. S. Golub & D. R. Gren, eds., Sinauer Associates,Sunderland, Mass., 1991, incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). Moreover,amino acids may be modified by glycosylation, phosphorylation and thelike.

To select candidate vaccine viruses according to the invention, thecriteria of viability, attenuation and immunogenicity are determinedaccording to well known methods. Viruses which will be most desired invaccines of the invention must maintain viability, have a stableattenuation phenotype, exhibit replication in an immunized host (albeitat lower levels), and effectively elicit production of an immuneresponse in a vaccinee sufficient to confer protection against seriousdisease caused by subsequent infection from wild-type virus. Therecombinant PIV of the invention are not only viable and moreappropriately attenuated than previous vaccine candidates, but are morestable genetically in vivo—retaining the ability to stimulate aprotective immune response and in some instances to expand theprotection afforded by multiple modifications, e.g., induce protectionagainst different viral strains or subgroups, or protection by adifferent immunologic basis, e.g., secretory versus serumimmunoglobulins, cellular immunity, and the like.

Recombinant PIV of the invention can be tested in various well known andgenerally accepted in vitro and in vivo models to confirm adequateattenuation, resistance to phenotypic reversion, and immunogenicity forvaccine use. In in vitro assays, the modified virus (e.g., a multiplyattenuated, biologically derived or recombinant PIV) is tested, e.g.,level of replication, for temperature sensitivity of virus replication,i.e. ts phenotype, and for the small plaque or other desired phenotype.Modified viruses are further tested in animal models of PIV infection. Avariety of animal models have been described and are summarized invarious references incorporated herein. PIV model systems, includingrodents and non-human primates, for evaluating attenuation andimmunogenic activity of PIV vaccine candidates are widely accepted inthe art, and the data obtained therefrom correlate well with PIVinfection, attenuation and immunogenicity in humans.

In accordance with the foregoing description, the invention alsoprovides isolated, infectious recombinant PIV viral compositions forvaccine use. The attenuated virus which is a component of a vaccine isin an isolated and typically purified form. By isolated is meant torefer to PIV which is in other than a native environment of a wild-typevirus, such as the nasopharynx of an infected individual. Moregenerally, isolated is meant to include the attenuated virus as acomponent of a cell culture or other artificial medium where it can bepropagated and characterized in a controlled setting. For example,attenuated PIV of the invention may be produced by an infected cellculture, separated from the cell culture and added to a stabilizer.

For vaccine use, recombinant PIV produced according to the presentinvention can be used directly in vaccine formulations, or lyophilized,as desired, using lyophilization protocols well known to the artisan.Lyophilized virus will typically be maintained at about 4° C. When readyfor use the lyophilized virus is reconstituted in a stabilizingsolution, e.g., saline or comprising SPG, Mg⁺⁺ and HEPES, with orwithout adjuvant, as further described below.

PIV vaccines of the invention contain as an active ingredient animmunogenically effective amount of PIV produced as described herein.The modified virus may be introduced into a host with a physiologicallyacceptable carrier and/or adjuvant. Useful carriers are well known inthe art, and include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, hyaluronic acid and the like. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration, as mentioned above. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, and the like. Acceptable adjuvants include incomplete Freund'sadjuvant, MPL™ (3-o-deacylated monophosphoryl lipid A; RIBI ImmunoChemResearch, Inc., Hamilton, Mont.) and IL-12 (Genetics Institute,Cambridge Mass.), among many other suitable adjuvants well known in theart.

Upon immunization with a PIV composition as described herein, viaaerosol, droplet, oral, topical or other route, the immune system of thehost responds to the vaccine by producing antibodies specific for PIVvirus proteins, e.g., F and HN glycoproteins. As a result of thevaccination with an immunogenically effective amount of PIV produced asdescribed herein, the host becomes at least partially or completelyimmune to PIV infection, or resistant to developing moderate or severePIV infection, particularly of the lower respiratory tract.

The host to which the vaccines are administered can be any mammal whichis susceptible to infection by PIV or a closely related virus and whichhost is capable of generating a protective immune response to theantigens of the vaccinizing strain. Accordingly, the invention providesmethods for creating vaccines for a variety of human and veterinaryuses.

The vaccine compositions containing the PIV of the invention areadministered to a host susceptible to or otherwise at risk for PIVinfection to enhance the host's own immune response capabilities. Suchan amount is defined to be a “immunogenically effective dose.” In thisuse, the precise amount of PIV to be administered within an effectivedose will depend on the host's state of health and weight, the mode ofadministration, the nature of the formulation, etc., but will generallyrange from about 10³ to about 10⁷ plaque forming units (PFU) or more ofvirus per host, more commonly from about 10⁴ to 10⁶ PFU virus per host.In any event, the vaccine formulations should provide a quantity ofmodified PIV of the invention sufficient to effectively protect the hostpatient against serious or life-threatening PIV infection.

The PIV produced in accordance with the present invention can becombined with viruses of other PIV serotypes or strains to achieveprotection against multiple PIV serotypes or strains. Alternatively,protection against multiple PIV serotypes or strains can be achieved bycombining protective epitopes of multiple serotypes or strainsengineered into one virus, as described herein. Typically when differentviruses are administered they will be in admixture and administeredsimultaneously, but they may also be administered separately.Immunization with one stain may protect against different strains of thesame or different serotype.

In some instances it may be desirable to combine the PIV vaccines of theinvention with vaccines which induce protective responses to otheragents, particularly other childhood viruses. In another aspect of theinvention the PIV can be employed as a vector for protective antigens ofother pathogens, such as respiratory syncytial virus (RSV) or measlesvirus, by incorporating the sequences encoding those protective antigensinto the PIV genome or antigenome which is used to produce infectiousPIV, as described herein (see, e.g., U.S. Provisional Patent ApplicationSer. No. 60/170,195, filed Dec. 10, 1999 by Murphy et al., incorporatedherein by reference).

In all subjects, the precise amount of recombinant PIV vaccineadministered, and the timing and repetition of administration, will bedetermined based on the patient's state of health and weight, the modeof administration, the nature of the formulation, etc. Dosages willgenerally range from about 10³ to about 10⁷ plaque forming units (PFU)or more of virus per patient, more commonly from about 10⁴ to 10⁶ PFUvirus per patient. In any event, the vaccine formulations should providea quantity of attenuated PIV sufficient to effectively stimulate orinduce an anti-PIV immune response, e.g., as can be determined bycomplement fixation, plaque neutralization, and/or enzyme-linkedimmunosorbent assay, among other methods. In this regard, individualsare also monitored for signs and symptoms of upper respiratory illness.As with administration to rhesus monkeys, the attenuated virus of thevaccine grows in the nasopharynx of vaccinees at levels approximately10-fold or more lower than wild-type virus, or approximately 10-fold ormore lower when compared to levels of incompletely attenuated PIV.

In neonates and infants, multiple administration may be required toelicit sufficient levels of immunity. Administration should begin withinthe first month of life, and at intervals throughout childhood, such asat two months, six months, one year and two years, as necessary tomaintain sufficient levels of protection against native (wild-type) PIVinfection. Similarly, adults who are particularly susceptible torepeated or serious PIV infection, such as, for example, health careworkers, day care workers, family members of young children, theelderly, individuals with compromised cardiopulmonary function, mayrequire multiple immunizations to establish and/or maintain protectiveimmune responses. Levels of induced immunity can be monitored bymeasuring amounts of neutralizing secretory and serum antibodies, anddosages adjusted or vaccinations repeated as necessary to maintaindesired levels of protection. Further, different vaccine viruses may beindicated for administration to different recipient groups. For example,an engineered PIV strain expressing a cytokine or an additional proteinrich in T cell epitopes may be particularly advantageous for adultsrather than for infants.

PIV vaccines produced in accordance with the present invention can becombined with viruses expressing antigens of another subgroup or strainof PIV to achieve protection against multiple PIV subgroups or strains.Alternatively, the vaccine virus may incorporate protective epitopes ofmultiple PIV strains or subgroups engineered into one PIV clone, asdescribed herein.

The PIV vaccines of the invention elicit production of an immuneresponse that is protective against serious lower respiratory tractdisease, such as pneumonia and bronchiolitis when the individual issubsequently infected with wild-type PIV. While the naturallycirculating virus is still capable of causing infection, particularly inthe upper respiratory tract, there is a very greatly reduced possibilityof rhinitis as a result of the vaccination and possible boosting ofresistance by subsequent infection by wild-type virus. Followingvaccination, there are detectable levels of host-engendered serum andsecretory antibodies which are capable of neutralizing homologous (ofthe same subgroup) wild-type virus in vitro and in vivo. In manyinstances the host antibodies will also neutralize wild-type virus of adifferent, non-vaccine subgroup.

Preferred PIV vaccine candidates of the invention exhibit a verysubstantial diminuition of virulence when compared to wild-type virusthat is circulating naturally in humans. The virus is sufficientlyattenuated so that symptoms of infection will not occur in mostimmunized individuals. In some instances the attenuated virus may stillbe capable of dissemination to unvaccinated individuals. However, itsvirulence is sufficiently abrogated such that severe lower respiratorytract infections in the vaccinated or incidental host do not occur.

The level of attenuation of PIV vaccine candidates may be determined by,for example, quantifying the amount of virus present in the respiratorytract of an immunized host and comparing the amount to that produced bywild-type PIV or other attenuated PIV which have been evaluated ascandidate vaccine strains. For example, the attenuated virus of theinvention will have a greater degree of restriction of replication inthe upper respiratory tract of a highly susceptible host, such as achimpanzee, or rhesus monkey, compared to the levels of replication ofwild-type virus, e.g., 10- to 1000-fold less. In order to further reducethe development of rhinorrhea, which is associated with the replicationof virus in the upper respiratory tract, an ideal vaccine candidatevirus should exhibit a restricted level of replication in both the upperand lower respiratory tract. However, the attenuated viruses of theinvention must be sufficiently infectious and immunogenic in humans toconfer protection in vaccinated individuals. Methods for determininglevels of PIV in the nasopharynx of an infected host are well known inthe literature.

Levels of induced immunity provided by the vaccines of the invention canalso be monitored by measuring amounts of neutralizing secretory andserum antibodies. Based on these measurements, vaccine dosages can beadjusted or vaccinations repeated as necessary to maintain desiredlevels of protection. Further, different vaccine viruses may beadvantageous for different recipient groups. For example, an engineeredPIV strain expressing an additional protein rich in T cell epitopes maybe particularly advantageous for adults rather than for infants.

In yet another aspect of the invention the PIV is employed as a vectorfor transient gene therapy of the respiratory tract. According to thisembodiment the recombinant PIV genome or antigenome incorporates asequence which is capable of encoding a gene product of interest. Thegene product of interest is under control of the same or a differentpromoter from that which controls PIV expression. The infectious PIVproduced by coexpressing the recombinant PIV genome or antigenome withthe N, P, L and other desired PIV proteins, and containing a sequenceencoding the gene product of interest, is administered to a patient.Administration is typically by aerosol, nebulizer, or other topicalapplication to the respiratory tract of the patient being treated.Recombinant PIV is administered in an amount sufficient to result in theexpression of therapeutic or prophylactic levels of the desired geneproduct. Representative gene products which may be administered withinthis method are preferably suitable for transient expression, including,for example, interleukin-2, interleukin-4, gamma-interferon, GM-CSF,G-CSF, erythropoietin, and other cytokines, glucocerebrosidase,phenylalanine hydroxylase, cystic fibrosis transmembrane conductanceregulator (CFTR), hypoxanthine-guanine phosphoribosyl transferase,cytotoxins, tumor suppressor genes, antisense RNAs, and vaccineantigens.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE I Construction of cDNAs Encoding a Chimeric HPIV3/BPIV3Antigenome and Recovery of Infectious Virus

The following three examples document studies to identify which of theproteins of BPIV3 contribute to its host range restriction in primates.To illustrate these methods, the N protein of the wild type HPIV3 viruswas replaced with its counterpart from BPIV3. This exchange wasaccomplished using a reverse genetics system for recovery of infectiousPIV from cDNA as described above. The studies were initiated with the Ngene of BPIV3 because this protein possesses an intermediate level ofamino acid sequence difference from its HPIV3 counterpart compared toother HPIV3 and BPIV3 proteins (see Example I).

A chimeric recombinant virus was constructed in which the N ORF of theJS strain of HPIV3 was replaced by that of either the Ka or SF strain ofBPIV3 (FIG. 1). These chimeric viruses possess the HN and Fglycoproteins of the HPIV3 parent and will induce a high level ofimmunity to HPIV3 in primates. Both chimeric viruses were successfullyrecovered. Both grew to high titer in cell culture and both were foundto be attenuated in rhesus monkeys. Thus, the N protein was identifiedas an exemplary protein that contributes to the host range phenotype ofBPIV3. Immunization of rhesus monkeys with either the Ka or SF chimericrecombinant virus induced a high level of resistance to the replicationof HPIV3 used as a wild type challenge.

The present invention, therefore, establishes the usefulness of reversegenetics methods to generate chimeric human-bovine PIV virus thatcombines the host range attenuation properties of BPIV3 and theimmunogenicity of the HPIV3 HN and F protective antigens. Immunizationof humans with such a chimeric recombinant will redress the problem ofsuboptimal immunogenicity of the BPIV3 vaccine previously observed inhumans.

The complete consensus nucleotide sequence for each of the Ka or SFBPIV3 strains was determined from RT-PCR products generated from virionRNA. These sequences are set forth in FIGS. 6A-6G, and FIGS. 7A-7G,respectively. The full length cDNA encoding a complete 15456 nucleotide(nt) antigenomic RNA of BPIV3 Ka is set forth in FIGS. 6A-6G herein (seealso GenBank accession #AF178654). The GenBank sequence for BPIV3 kansasstrain differs from the sequence of the exemplary cDNA in two positionsat nucleotide 21 and 23. Both, the published sequence and the sequencein the exemplary cDNA occur naturally in kansas strain virus populationwith similar frequencies. The cDNA used in the present example containsa sequence beginning at nucleotide 18, ACTGGTT (SEQ [D NO. 1), whereasthe corresponding published sequence (GenBank accession #AF178654; FIGS.6A-6G, SEQ ID NO. 22) reads ACTTGCT (differing nucleotides at positions21 and 23 are underscored).

To construct consensus nucleotide sequences for the Ka and SF BPIV3strains, virion RNA was subjected to reverse transcription using theSuperscript II Preamplification System (Life Technologies, Gaithersburg,Md.) and 200 ng of random hexamer primers. PCR was carried out on thefirst strand product using the Advantage cDNA PCR kit (ClontechLaboratories, Palo Alto, Calif.). Ka and SF genomes were each amplifiedby PCR in 3 or 4 overlapping fragments using primers homologous toregions of RNA conserved among previously-published paramyxovirussequences. Each primer pair was constructed to include matchingrestriction enzyme sites (not represented in the sequence targeted foramplification).

A separate random library was generated for each amplicon by digesting aset of PCR products with the appropriate restriction enzyme, followed bygel-purification, ligation of the products into tandem arrays andsonication. A random library was generated from this pool of shearedcDNA sequences by cloning a subset (approx. 500 bp fragments) into M 13.The nucleotide sequences of cDNA inserts were determined by automatedDNA sequencing using the Taq DYE Deoxy Terminator cycle sequencing kit(ABI, Foster City, Calif.). A continuous sequence (contig) was assembledfor each of the original large RT-PCR fragments with sufficientredundancy that each nucleotide position was confirmed by a minimum 3independent M13 clones. The 5′ and 3′ terminal genomic sequences of Kaand SF were converted to cDNA using the system for Rapid Amplificationof cDNA Ends (Life Technologies, Gaithersburg, Md.) and sequenced byautomated sequencing.

These sequences are set forth in FIGS. 6A-6G (Ka) and FIGS. 7A-7G (SF),respectively. Analysis of these sequences revealed that the percentamino acid identity between HPIV3 and BPIV3 for each of the followingproteins is: N (86%), P (65%), M (93%), F (83%), HN (77%), and L (9 1%).Thus sequence divergence was found distributed over many genes. Thededuced amino acid sequence of the N genes of these two viruses ispresented in GenBank #Af178654 (Ka) and #AF178655 (SF), not included.The position of the N ORF in the BPIV3 genome is indicated in therespective BenBank reports and included herein by reference. In theexample below, the N ORF of the Ka or SF virus was initially selectedfor replacement of the corresponding gene in the HPIV3 virus because theN gene represents a gene with an intermediate level of sequencedivergence among the six HPIV3 and BPIV3 proteins. In this study the NORF, but not the 3′ or 5, noncoding N gene sequences, was exchanged,which permitted us to assign an observed attenuation phenotype of cKaand cSF to the protein encoded by the N gene.

Human-bovine chimeric full-length PIV3 genomes were constructed byintroducing the BPIV3 Ka or SF N coding region as a replacement for itsHPIV3 counterpart into the rJS cDNA p3/7(131)2G which encodes a completecopy of HPIV3 positive-sense antigenomic RNA (see, e.g., Durbin et al.,1997a, supra; Hoffman et al., 1997, supra; Skiadopoulos et al., 1998,supra; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998;U.S. Provisional Application No. 60/047,575, filed May 23, 1997(corresponding to International Publication No. WO 98/53078), and U.S.Provisional Application No. 60/059,385, filed Sep. 19, 1997; eachincorporated herein by reference). BPIV3 and HPIV3 N coding regions withflanking sequences were first subcloned and further modified to permitan exchange of just the N ORF. pUC119JSN bearing the HPIV3 N gene andthe plasmids with a BPIV3 N Ka or SF gene (pBSKaN and _(P)BSSFN) weresubjected to mutagenesis using the method of Kunkel (Proc. Natl. Acad.Sci. USA 82:488-492, 1985, incorporated herein by reference) tointroduce Ncol and AflII restriction enzyme recognition sites attranslational start and stop sites, respectively (FIG. 1A). FollowingNcol/AflII digestion of pUC119KaN-Ncol/AflII, the BPIV3 N coding regionwas introduced as an Ncol/AflII fragment into pUC119JSN-Ncol/AflII as areplacement for the HPIV3 N coding region (FIG. 1B). The chimeric Ngenes, which contain the HPIV3 3′ and 5′ noncoding sequences and theBPIV3 ORF, were modified by site-directed mutagensis to restore theoriginal HPIV3 noncoding sequence and BPIV3 coding sequence. Thischimeric N gene was then introduced into the 5′ half of the rJSantigenome, pLeft, in exchange for its corresponding HPIV3 sequence(FIGS. 2A and 2B) using existing MIul and EcoRI sites present in thehuman sequence. In each case parallel reactions were carried out for theSF N ORF. The chimeric pLeft plasmid was combined with the Xhol/NgoMlfragment from pRight containing the 3′ half of the rJS antigenomeflanked by the delta ribozyme and the T7 terminator at its 3′ end (FIG.2). The resulting chimeric PIV3 plasmids designated pB/HPIV3NKa orpB/HPIV3NSF, contained the full-length rJS antigenome in which the N ORFencoded the BPIV3 Ka or SF N protein.

Chimeric antigenomic HPIV3/BPIV3 cDNAs were transfected individuallyinto HEp-2 cells grown to near-confluence in 6-well plates along withtwo previously-described support plasmids, pTM(P no C) and pTM(L),Lipofectace (Life Technologies, Gaithersburg, Md.), and infected with amodified vaccinia virus recombinant that expresses bacteriophage T7 RNApolymerase (MVA-T7) as previously described (Durbin et al., Virology234:74-83, 1997b). An N support plasmid used in previous work wasomitted because the antigenomic plasmid expressed sufficient levels ofthe N protein. The cultures were maintained for 3.5 days at 32° C. afterwhich supernatants were harvested, passaged in LLC-MK2 cells andplaque-purified 3 times in LLC-MK2 cells. The identities of the chimericviruses incorporating a human PIV 3 background genome or antigenome anda BPIV3 N protein (designated as rHPIV3-N_(B) chimeric recombinants or,more specifically, as “cKa” and “cSF” chimeric viruses) recovered fromthe transfections were confirmed by sequencing RT-PCR productscontaining the regions of the N ORF start and stop codons from virionRNA isolated after amplification of triply plaque-purified virus (FIG.3). This amplified product and the corresponding amplified HPIV3 rJS andBPIV3 Ka or SF sequences were also subjected to TaqI digestion toconfirm the chimeric identity of cKa and cSF viruses (FIG. 4). TaqIdigestion profiles were distinct for the 3 parental and 2 chimericviruses, and each parental profile included TaqI fragments of uniquesize, allowing the contribution of sequence of rJS, Ka and SF parents tothe chimeric viruses to be verified. The recovered cKa and cSF chimericrecombinants each contained the expected sequences as designed.

EXAMPLE II Replication of HPIV3/BPIV3 Chimeric Viruses in Cell Culture

Efficient replication of live attenuated virus vaccines in tissueculture cells is a feature of human-bovine chimeric PIV of the inventionthat permits efficient manufacture of the recombinant vaccine materials.The multicycle replication of rJS parent, cKa, Ka parent, cSF, and SFparent in a bovine cell line (MDBK) and in a simian cell line (LLC-MK2)was determined by infecting cells with virus at a multiplicity ofinfection of 0.01 and harvesting samples (in triplicate) over a five dayperiod of time (FIG. 5) as previously described (Tao et al., 1998,supra, incorporated herein by reference). The chimeric virusesreplicated efficiently in both cell lines like their human or bovineparent viruses without significant delay in replication or a significantreduction in the titer of virus achieved. In each case, the chimericviruses replicated to over 10^(7.0)TCID₅₀/ml which is well above the10^(4.0) or 10^(5.0) dose of live attenuated human or bovine PIVvaccines currently being used in human clinical trials (Karron et al.,1996, supra; Karron et al., 1995a, supra; and Karron et al., 1995b,supra).

EXAMPLE III Evaluation of Attenuation and Protective Efficacy of theHPIV3/BPIV3 Chimeric Viruses in Rhesus Monkeys

Both the SF and Ka BPIV3s are attenuated for the upper and the lowerrespiratory tract of the rhesus monkey (van Wyke Coelingh et al., 1988,supra). This attenuation phenotype correlates with attenuation in humans(Karron et al., 1995a, supra) as indicated by the fact that Ka is highlyrestricted in replication in the upper respiratory tract of fullysusceptible seronegative infants and children. The absence of cough,croup, bronchiolitis, or pneumonia in the BPIV3-infected vaccineessuggests that the Ka BPIV3 virus is attenuated for the lower respiratorytract as well. Therefore, the rhesus monkey is widely accepted as areasonably correlative model to evaluate attenuation of candidate PIVvaccine viruses and their efficacy against challenge with wild type PIV.

The rJS, cKa, Ka parent, cSF, and SF parent were administeredintranasally and intratracheally at a dose of 10^(5.0) TCID₅₀ per siteto rhesus monkeys. Replication was monitored using previously describedprocedures for obtaining samples from the upper (nasopharyngeal swabspecimens) and lower (tracheal lavage specimens) respiratory tract andfor titering the virus in LLC-MK2 cells (Hall et al., 1992, supra). ThecKa and cSF recombinants were significantly attenuated for the upperrespiratory tract (Table 1) exhibiting, respectively, a 63-fold or a32-fold reduction in mean peak virus titer compared to that of the rJSHPIV3 parent. Both cKa and cSF were also attenuated for the lowerrespiratory tract, but this difference was only statisticallysignificant for cSF. The low level of replication of rJS in the lowerrespiratory tract made it difficult to demonstrate in astatistically-significant fashion further restriction of replication dueto an attenuation phenotype at this site.

TABLE 1 Replication of cKa and cSFis restricted relative to HPIV3 in theupper and lower respiratory tracts of rhesus monkeys. Virus ReplicationMean titers Mean peak titers Log₁₀TCID₅₀/ml ± standard error [Duncangrouping]² Log₁₀TCID₅₀/ml ± standard Immunizing No. of NasopharynxTrachea error [Duncan grouping] virus¹ animals day 6 day 7 day 4 day 6Nasopharynx Trachea rJS 4 5.3 ± 0.59[A] 3.9 ± 036[A] 1.7 ± 0.45[A] 1.7 ±0.29[A] 5.3 ± 0.59[A] 2.5 ± 50.51[A] cKa 4 3.0 ± 0.58[B] 2.9 ± 0.42[AB]1.5 ± 0.40[A] 1.0 ± 0.19[A] 3.5 ± 0.54[B] 1.5 ± 0.18[AB] Ka 4 2.0 ±0.27[B] 2.4 ± 0.30[B] 1.3 ± 0.26[A] 1.3 ± 0.21[A] 2.5 ± 0.30[B] 1.6 ±0.15[AB] cSF 4 3.3 ± 0.40[B] 3.7 ± 0.57[A] 1.1 ± 0.25[A] 1.1 ± 0.24[A]3.8 ± 0.46[B] 1.4 ± 0.26[B] SF 4 2.8 ± 0.48[B] 2.6 ± 0.40[AB] 1.6 ±0.46[A] 1.5 ± 0.40[A] 3.3 ± 0.28[B] 1.8 ± 0.41[AB] ¹Monkeys wereinoculated intranasally and intratracheally with 10^(5.0)TCID⁵⁰ in 1 mlat each site. ²Mean viral titers in each column were assigned tostatistically similar groups (designated with a letter) using a DuncanMultiple Range test (α = 0.05). Mean titers in each column withdifferent letters are statistically different.

The level of replication of each chimeric virus, cKa and cSF, was notsignificantly different from its bovine parent in the upper or the lowerrespiratory tract, although the chimeric viruses each replicatedsomewhat better than their BPIV3 parents in the upper respiratory tract.Thus, the acquisition of the N gene of either the Ka or SF BPIV3 by rJSHPIV3 attenuated the human virus for rhesus monkeys to a levelapproximately equivalent to that of the BPIV parent. Since theHPIV3/BPIV3 chimeric recombinants replicated efficiently in tissueculture cells in vitro, it is clear that the phenotype of host rangerestricted replication manifested by the two bovine parental viruses wastransferred to HPIV3 by the N ORF. It is possible, but unknown andunpredictable, that substitution of other BPIV3 genes, such as M, P, orL, for their HPIV3 counterpart in rJS will give similar or greaterlevels of attenuation as observed upon substitution of the BPIV3 N genefor the HPIV3 N gene. The observation that the level of replication ofcKa and cSF is slightly greater than that of their BPIV3 parents in theupper respiratory tract suggests that additional bovine genes contributeto the host range attenuation phenotype of the BPIV3 parent virus atthis site.

Uninoculated monkeys and monkeys that were previously infected with ahuman or bovine PIV3 parental virus, or with the cKa or cSF chimericvirus, were challenged 42 days after the initial inoculation with10^(6.0) TCID₅₀ of rJS intranasally and intratracheally in a 1 mlinoculum at each site. The nasopharynx and the trachea were sampled asdescribed previously on the days indicated in Table 2. The titer ofvirus present at each site was determined for each monkey on LLC-MK2cell monolayers, and the titers presented are mean peak titers (Hall etal., 1992, supra). Previous infection with either chimeric virus induceda high level of resistance to replication of the rJS challenge virus inboth the upper and lower respiratory tract. Monkeys previously infectedwith cKa manifested a 300-fold reduction of replication of wild typeHPIV3 (rJS) in the upper respiratory tract and a 1000-fold reduction inthe lower tract compared to uninoculated control monkeys. Monkeyspreviously infected with cSF manifested a 2000-fold reduction ofreplication of rJS in the upper respiratory tract and a 1000-foldreduction in the lower tract compared to uninoculated control monkeys.The level of reduction of replication of rJS challenge virus in monkeyspreviously-inoculated with cKa or cSF was comparable to that of monkeyspreviously infected with either the bovine or the human PIV parent.Thus, infection with either HPIV3/BPIV3 chimeric virus provided a highlevel of protection in the upper and lower respiratory tract of monkeys,and both chimeric viruses represent promising vaccine candidates.

Serum collected from monkeys on days 0 and 28 was tested by HAI assayusing HPIV3 (JS strain) and BPIV3 (Ka strain) as antigen as previouslydescribed (Coelingh et al., J. Infect. Dis. 157:655-662, 1988). AlthoughcKa-N and cSF-N were highly attenuated in the upper and lowerrespiratory tract of rhesus monkeys relative to rJS, each chimeric virusinduced a hemagglutination-inhibiting (HAI) antibody response to HPIV3that was 2.5 to 5-fold greater in magnitude than that induced byimmunization with its respective BPIV3 patent. This likely is due to thepresence of HPIV3 HN protein in the chimeric viruses. Furthermore, theHPIV3-specific HAI-responses induced by the chimeric viruses werestatistically indistinguishable from that induced by immunization withrJS. An additional unexpected result demonstrated herein is that,following challenge of the monkeys with HPIV3, the level of HAI antibodyin monkeys initially immunized with cKa-N or cSF-N was significantlygreater than levels observed in animals immunized with rJS, Ka or SF.

EXAMPLE IV Construction and Characterization of Chimeric HPIV3/BPIV3Vaccine Candidates Having Heterologous Fusion AndHemagglutinin-Neuraminidase Glycoproteins

In the preceding example, the basis of host range restriction ofreplication of BPIV3 for the respiratory tract of primates was examinedby the generation and characterization of a recombinant human PIV3(rHPIV3) in which the N open reading frame (ORF) was replaced by that ofits BPIV3 counterpart. The resulting chimeric virus, rHPIV3-N_(B), alsoreferred to as cKa or cSF, efficiently replicated in vitro but wasrestricted in replication in the upper respiratory tract of rhesusmonkeys, identifying the N protein as an independent determinant of thehost range restriction of BPIV3 in rhesus monkeys (Bailly et al., J.Virol. 74:3188-3195, 2000).

In the present example, the contribution of the fusion (F) andhemagglutinin-neuraminidase (HN) glycoprotein genes of bovineparainfluenza virus type 3 (BPIV3) to its restricted replication in therespiratory tract of non-human primates was examined by generating andcharacterizing two reciprocal chimeric BPIV3/HPIV3 viruses. A chimericHPIV3 containing heterologous BPIV3 F and HN glycoprotein genes in placeof its own, and the reciprocal recombinant comprising a BPIV3 “backbone”bearing the HPIV3 F and HN genes substituted for the counterpart BPIV3glycoprotein genes, were generated to assess the effect of glycoproteinsubstitution on replication of HPIV3 and BPIV3 in the upper and lowerrespiratory tract of rhesus monkeys. Thus, in one chimeric virus, the Fand HN genes of HPIV3 were replaced with their BPIV3 counterparts,resulting in a chimeric recombinant designated rHPIV3-F_(B)HN_(B). Thereciprocal chimeric recombinant PIV3 (rBPIV3-F_(H)HN_(H)) wasconstructed by replacing the F and HN genes of a recombinant BPIV3(rBPIV3) with their HPIV3 counterparts. In the latter virus, theintroduction of the HPIV3 F and HN ORFs into the BPIV3 backbone combinesthe antigenic determinants of HPIV3 with the backbone of BPIV3 and thusprovides an improved vaccine candidate compared with parental BPIV3. TheF and HN genes were exchanged as pairs in view of the proposedrequirement for homologous HN and F proteins for parainfluenza virusesfor full functional activity (Deng et al., Virology 209:457-469, 1995;and Tanabayashi et al., J. Virol. 70:6112-6118, 1996; each incorporatedherein by reference).

The foregoing chimeric viruses were readily recovered and exhibitedkinetics of replication in simian LLC-MK2 cells that were comparable tothose of their parent viruses, suggesting that the heterologousglycoproteins were compatible with the PIV3 internal proteins. Thedistinctive features of cytopathology of BPIV3 versus HPIV3 cosegregatedwith their respective F and HN genes. HPIV3 bearing the BPIV3 F and HNgenes was attenuated for replication in rhesus monkeys to a levelsimilar to that of its BPIV3 parent virus, indicating that theglycoprotein genes of BPIV3 are major determinants of its host rangerestriction of replication in rhesus monkeys. BPIV3 bearing the HPIV3 Fand HN genes (rBPIV3-F_(H)HN_(H)) replicated in rhesus monkeys to alevel intermediate between that of HPIV3 and BPIV3.

These results indicate that the F and HN genes make a significantcontribution to the overall attenuation of BPIV3. Furthermore, theydemonstrate that BPIV3 sequences outside the F and HN region alsocontribute to the attenuation phenotype in primates. This latter findingis consistent with the demonstration in the preceding example that thenucleoprotein coding sequence of BPIV3 is a determinant of itsattenuation for primates. Despite its restricted replication in therespiratory tract of rhesus monkeys, rBPIV3-F_(H)HN_(H) conferred alevel of protection against challenge with wild type HPIV3 that wasindistinguishable from that conferred by previous infection with wildtype HPIV3. From these and related findings, the usefulness ofrBPIV3-F_(H)HN_(H) as a vaccine candidate against HPIV3 is readilyapparent.

Viruses and Cells

HEp-2 and simian LLC-MK2 monolayer cell cultures were maintained-in MEMmedium (Life Technologies, Gaithersburg, Md.) supplemented with 5% fetalbovine serum (Summit Biotechnology, Ft. Collins, Colo.), 50 ug/mlgentamicin sulfate, and 4 mM glutamine (Life Technologies, Gaithersburg,Md.).

The wild type BPIV3 strain Kansas/15626/84 (Clone 5-2-4, Lot BP13-1)(BPIV3 Ka), the HPIV3 JS wild type, its recombinant version (rBPIV3),and the rHPIV3 virus containing the BPIV3 Ka N ORF in place of theHPIV3-N ORF (rHPIV3-N_(B)) are each described above (see also, Clementset al., 1991, supra; Karron et al., 1995a, supra; Bailly et al., 2000,supra; and Durbin et al., 1997, supra). PIVs were propagated at 32° C.in LLC-MK2 cells (ATCC CCL-7), as previously described (Hall et al.,1992, supra). The modified vaccinia strain Ankara (MVA) recombinantvirus that expresses bacteriophage T7 RNA polymerase is described byWyatt et al. (1995, supra).

Construction of Antigenomic cDNAs Encoding Recombinant BPIV3/HPIV3Viruses.

a) Construction of cDNA to Recover rBPIV3

A full length cDNA was constructed to encode the complete 15456nucleotide (nt) antigenomic RNA of BPIV3 Ka, as described above. ThecDNA was assembled from 4 subclones derived from reverse transcription(RT) of viral RNA using the SuperScript II Pre-amplification System(Life Technologies, Gaithersburg, Md.) and polymerase chain reaction(PCR) amplification with a High Fidelity PCR kit (Clontech Laboratories,Palo Alto, Calif.) The RT-PCR products were cloned into modified pUC19plasmids (New England Biolabs, Beverly, Mass.) using the followingnaturally occuring internal restriction enzyme recognition sites: Sma I(BPIV3 Ka sequence position nt186), Pst I (nt 2896), Mlu I (nt 6192),Sac II (nt 10452) and Bsp LU11 (nt 15412). Multiple subclones of theantigenomic cDNA were sequenced using a Perkin Elmer ABI 310 sequencerwith dRhodamine Terminator Cycle Sequencing (Perkin Elmer AppliedBiosystems, Warrington, UK), and only those matching the consensussequence of BPIV3 Ka were used for assembly of the full length clone.The 3′ and 5′ ends of BPIV3 Ka were cloned and the assembly of the fulllength cDNA took place in the previously described p(Right) vector(Durbin et al., 1997, supra), which we modified to contain a newpolylinker with restriction enzyme recognition sites for Xho I, Sma I,Mlu I, Sac II, Eco RI, Hind III and RsrII. The full length cDNA clonepBPIV3(184) contained the following elements in 3′ to 5′ order: a T7promoter followed by 2 non-viral guanosine residues, the completeantigenomic sequence of BPIV3 Ka, a hepatitis delta virus ribozyme and aT7 polymerase transcription terminator (Bailly et al., 2000, supra; andDurbin et al., 1997a, supra).

b) Construction of rHPIV3-F_(B)HN_(B) and rBPIV3-F_(H)HN_(H)

Unique restriction enzyme recognition sites were introduced into theBPIV3 antigenomic cDNA and into the previously described HPIV3antigenomic cDNA p3/7(131)2G (Durbin et al., 1997a, supra) to facilitatethe exchange of the F and HN genes between BPIV3 and HPIV3 cDNAs. Usingthe transformer site-directed mutagenesis protocol from Clontech(Clontech Laboratories, Palo Alto, Calif.), SgrAI restriction sites wereintroduced in the downstream non-coding region of the M gene at position4811 of the rBPIV3 sequence and position 4835 of the rHPIV3 JS sequence(GenBank accession # Z11575). The nucleotide number given for theposition of restriction enzyme recognition sites indicates thenucleotide after which the enzyme cuts, not the first nucleotide of therestriction enzyme recognition site. The sequence was changed fromTCCAACATTGCA (SEQ. ID. NO. 2) to TCCACCGGTGCA (SEQ. ID. NO. 3) in rBPIV3and from CGGACGTATCTA (SEQ. ID. NO. 4) to CGCACCGGTGTA (SEQ. ID. NO. 5)in rHPIV3 (recognition sites underlined). BsiWI restriction sites wereintroduced in the downstream non-coding region of the HN gene at nt 8595of the rBPIV3 sequence and at nt 8601 of the rHPIV3 JS sequence. Thesequence was changed from GATATAAAGA (SEQ. ID. NO. 6) to GACGTACGGA(SEQ. ID. NO. 7) in rBPIV3 to give pBPIVs(107) and from GACAAAAGGG (SEQ.ID. NO. 8) to GACGTACGGG (SEQ. ID. NO. 9) in rHPIV3 to give pHPIVs(106).The F and HN genes were exchanged between pBPIVs(107) and pHPIV3s(106)by digestion of each with SgrAI and BsiWI, gel purification of thefragments, and assembly of the appropriate fragments into the two fulllength cDNAs. The HPIV3 backbone bearing the BPIV3 F and HN genes,designated pHPIV(215), encoded 15480 nts of viral sequence, of which nts4835 to 8619 came from BPIV3, and it was used to deriverHPIV3-F_(B)HN_(B) (FIGS. 8A-8C). The BPIV3 backbone bearing the HPIV3 Fand HN genes, designated pBPIV(215), encoded 15438 nts of viralsequence, of which nts 4811 to 8577 came from HPIV3, and it was used toderive rBPIV3-F_(H)HN_(H) (FIGS. 8A-8C).

BPIV3 Support Plasmids for Recovery of Virus from cDNA.

Support plasmids encoding the BPIV3 Ka N, P and L genes were assembledin modified pUC19 vectors and then cloned into the previously describedpTM-1 vector (Durbin et al., 1997a, supra). In order to place theindividual genes immediately downstream of the T7 promoter in the pTMvector, an Nco I site was introduced at the start codon of the N, P andL open reading frames (ORFs) using site-directed mutagenesis. The Nco Irestriction site and a naturally occurring restriction site downstreamof each ORF (Spe I for N, HincII for P and Bsp LU11I for L) was used forcloning into pTM. After cloning, the Nco I site in pTM(N) wasmutagenized back to the original sequence to restore the correct aminoacid assignment in the second codon. In pTM(P) and pTM(L) the amino acidsequence encoded by the ORF was not altered by the introduction of Nco Isites.

Transfection.

HEp-2 cells (approximately 1.5×10⁶ cells per well of a six-well plate)were grown to 90% confluence and transfected with 0.2 μg each of theBPIV3 support plasmids pTM(N) and pTM(P), and 0.1 μg of pTM(L), alongwith 5 μg of the fill length antigenomic cDNA and 12 μl LipofectACE(Life Technologies, Gaithersburg, Md.). Each transfection mixture alsocontained 1.5×10⁷ plaque forming units (PFU) of MVA-T7, as previouslydescribed (Durbin et al., 1997, supra). The cultures were incubated at32° C. for 12 hrs before the medium was replaced with MEM (LifeTechnologies, Gaithersburg, Md.) containing 10% fetal bovine serum. Thesupernatants were harvested after incubation at 32° C. for an additionalthree days, and were passaged onto LLC-MK2 cell monolayers in 25 cm²flasks and incubated for 5 days at 32° C. Virus present in thesupernatant was plaque-purified three times prior to amplification andcharacterization.

Molecular Characterization of Recovered Chimeric Recombinants.

The presence of the heterologous F and HN genes in the bovine or humanPIV3 backbone was confirmed in plaque-purified recombinant viruses byRT-PCR of viral RNA isolated from infected cells or supernatant, whichwas performed using a primer pair that recognizes conserved sequences inrBPIV3 and rHPIV3. This yielded similarly sized fragments (nts 4206-9035in rBPIV3, nts 4224-9041 in rHPIV3, nts 4206-9017 in rBPIV3-F_(H)HN_(H),and nts 4224-9059 in rHPIV3-F_(B)HN_(B)) which were then digested withEco RI and analyzed by electrophoresis on a 1% agarose gel (FIG. 9). Thenucleotide sequence flanking the introduced SgrAI and BsiWI restrictionsites in each virus was confirmed by sequencing the respective RT-PCRproduct.

Replication of HPIV3/BPIV3 Chimeric Viruses in Cell Culture.

The multicycle growth kinetics of BPIV3 Ka, rHPIV3-F_(B)HN_(B),rBPIV3-F_(H)HN_(H), rHPIV3-N_(B) and rHPIV3 in LLC-MK2 cells weredetermined by infecting cells in triplicate at a multiplicity ofinfection (MOI) of 0.01 and harvesting samples at 24 hr intervals over asix day period, as previously described (Tao et al., 1998, supra).Samples were flash-frozen and titered in a single assay on LLC-MK2 cellmonolayers in 96 well plates at 32° C., as described (Durbin et al.,Virology 261:319-330, 1999b, incorporated herein by reference).

Primate Model Studies.

Rhesus monkeys seronegative for PIV3 as determined byhemagglutination-inhibition (HAI) assay (van Wyke Coelingh et al., 1988,supra) were inoculated intranasally and intratracheally in groups of 2or 4 animals with 10⁵ tissue culture infectious dose₅₀ (TCID₅₀) per mlof BPIV3 Ka, rHPIV3-F_(B)HN_(B), rBPIV3-F_(H)HN_(H), rHPIV3-N_(B) orrHPIV3. Nasopharyngeal swabs were collected daily on days 1 to 11 and onday 13. Tracheal lavage samples were collected on days 2, 4, 6, 8, and10 post-infection. Individual samples were flash-frozen and stored at−70° C. until all samples were available for titration. Virus in thespecimens was titered on LLC-MK2 cell monolayers in 24 and 96 wellplates as previously described (Durbin et al., 1999b, supra). Seracollected from monkeys on days 0 and 28 was tested by HAI assay usingHPIV3 JS and BPIV3 Ka as antigens, as previously described (van WykeCoelingh et al., 1988, supra). On day 28 post inoculation, the monkeyswere challenged intranasally and intratracheally with 10⁶TCID₅₀ per siteof HPIV3 JS. Nasopharyngeal swab samples were collected on days 3, 4, 5,6, 7 and 8, and tracheal lavage samples on days 4, 6 and 8 postchallenge. Samples were titered in a single assay as described above.Serum was collected on day 28 post challenge.

Recovery of rBPIV3 and BPIV3/HPIV3 Chimeric Viruses (rHPIV3-F_(B)HN_(B)and rBPIV3-F_(H)HN_(H)) from cDNA.

A complete BPIV3 antigenomic cDNA, designated pBPIV(184), wasconstructed to encode the consensus sequence of BPIV3 Ka. This BPIV3antigenomic cDNA was further modified by the introduction of uniqueSgrAI and BsiWI sites into the downstream noncoding region of the M andHN genes, respectively (FIG. 8C). The same restriction sites wereintroduced into the downstream noncoding region of the M and HN genes ofa previously described complete HPIV3 antigenomic cDNA, p3/7(131)2G(Durbin et al., 1997a, supra). The F and HN glycoprotein genes of HPIV3and BPIV3 were swapped by exchanging this SgrAI-BsiWI restrictionfragment. A direct exchange of entire genes was anticipated to bewell-tolerated because of the high level of sequence conservationbetween the cis-acting signals of BPIV3 and HPIV3. The HPIV3 antigenomiccDNA bearing the BPIV3 F and HN genes was designated pHPIV(215), and theBPIV3 antigenomic cDNA bearing the HPIV3 F and HN genes was designatedpBPIV(215).

The antigenomic cDNAs pBPIV(184), pHPIV(215), pBPIV(215) and p3/7(131)2Gwere separately transfected into HEp-2 cells along with the three BPIV3support plasmids pTM(N), pTM(P) and pTM(L), and the cells weresimultaneously infected with recombinant MVA expressing the T7 RNApolymerase. To confirm that the recovered viruses indeed were theexpected rBPIV3, rHPIV3-F_(B)HN_(B), rBPIV3-F_(H)HN_(H) and rHPIV3viruses, intracellular RNA or RNA from supernatant from each clonedvirus was analyzed by RT-PCR using a primer pair that recognizedidentical sequences in HPIV3 JS and BPIV3 Ka. The primer pair amplifieda 4.8 kb fragment of DNA corresponding to the downstream end of the Mgene, the F and HN genes, and the upstream end of the L gene (nts4206-9035 in rBPIV3, nts 4224-9041 in rHPIV3, nts 4206-9017 inrBPIV3-F_(H)HN_(H,) and nts 4224-9059 in rHPIV3-F_(B)HN_(B)). Thegeneration of each PCR product was dependent upon the inclusion ofreverse transcriptase, indicating that each was derived from viral RNAand not from contaminating cDNA (data not shown). The PCR products werethen digested with Eco R1, which would be predicted to yield adifferent, unique restriction enzyme digest pattern for each of the fourviruses (FIG. 9). In each case, the predicted pattern was observed,confirming the identity of the backbone and the inserted F and HN genes.In addition, nucleotide sequencing was performed on the RT-PCR productsto confirm the presence of the introduced restriction sites and flankingsequences.

The cytopathic effect (CPE) caused by rBPIV3-F_(H)HN_(H) in LLC-MK2cells was indistinguishable from that of HPIV3 JS (condensed, rounded-upcells and small syncytia) but different from BPIV3 (large multicellularsyncytia), whereas the CPE caused by rHPIV3-F_(B)HN_(B) was identical tothat caused by the BPIV3. This indicates that the cytopathology of thechimeric PIVs cosegregated with the parental origin of the F and HNgenes.

BPIV3/HPIV3 Chimeric Viruses Replicate Efficiently in Cell Culture.

The growth kinetics of rHPIV3-F_(B)HN_(B) and rBPIV3-F_(H)HN_(H) werecompared with that of their parental viruses by infecting LLC-MK2monolayers at an MOI of 0.01 and monitoring the production of infectiousvirus. The kinetics and magnitude of replication of the two chimericviruses were comparable to those of their HPIV3 or BPIV3 parentalviruses (FIG. 10). This suggested that BPIV3 and HPIV3 glycoproteinswere compatible with the heterologous PIV3 internal proteins. This is animportant property because it will be possible to efficiently preparevaccine virus.

The F and HN Genes of the BPIV3/HPIV3 Chimeric Viruses are Determinantsof the Host Range Restriction of Replication of BPIV3 Ka in theRespiratory Tract of Rhesus Monkeys.

rHPIV3-F_(B)HN_(B) and rBPIV3-F_(H)HN_(H) were evaluated for theirability to replicate in the upper and lower respiratory tract of rhesusmonkeys. In particular, the effects of introduction of the BPIV3 F andHN genes into HPIV3 on attenuation of replication in rhesus monkeys wasdemonstrated, as described above for the BPIV3 N protein (see also,Bailly et al., 2000, supra). In addition, the effects of introduction ofthe HPIV3 F and HN genes into BPIV3 on replication in rhesus monkeys wasdetermined. If the predominant attenuating mutations of BPIV3 were ingenes other than the F and HN, then one would expect little overalleffect of the HPIV3-BPIV3 glycoprotein exchange on replication of BPIV3in rhesus monkeys.

Each chimeric virus was administered intranasally and intratracheally torhesus monkeys at a dose of 10⁵ TCID₅₀ per site. The level ofreplication of the chimeric viruses was compared to that of the rHPIV3and BPIV3 parental viruses and to that of rHPIV3-N_(B) (Table 3). Sincethe rHPIV3 parental virus replicated to a low to moderate level in thelower respiratory tract, meaningful comparisons between groups couldonly be made for replication in the upper respiratory tract. The levelof replication of rHPIV3-F_(B)HN_(B) was similar to that of its BPIV3parent and substantially lower than that of its HPIV3 parent (Table 3;FIG. 11, panel A). This showed that the BPIV3 glycoprotein genescontained one or more major determinants of the host range attenuationphenotype of BPIV3 for rhesus monkeys. The magnitude and pattern ofreplication of rHPIV3-F_(B)HN_(B) and rHPIV3-N_(B) were very similar,indicating that each of the two bovine genetic elements, namely the Ngene versus the F and HN genes, attenuate HPIV3 to a similar extent.

TABLE 3 The F and HN glycoprotein genes of BPIV3 contribute to itsrestricted replication in the respiratory tract of rhesus monkeys. Meanpeak virus titer³ Serum HAI antibody titer Serum HAI antibody(log₁₀TCID₅₀/ml ± S.E.) (mean recip. titer (mean recip. ImmunizingNumber [Duncan Grouping]⁴ log₂ ± S.E.) for log₂ ± S.E.) for virus¹ ofanimals² NP swab⁵ Tracheal lavage⁶ HPIV3 on day 28⁷ BPIV3 on day 28⁷rHPIV3 6 4.7 ± 0.54 [A] 2.4 ± 0.37 [A] 9.5 ± 0.72 [A] 6.8 ± 1.03 [B]rBPIV3-F_(H)HN_(H) 4 3.1 ± 0.58 [B] 1.6 ± 0.05 [A] 6.8 ± 0.63 [BC] 3.8 ±0.63 [C] rHPIV3-N_(B) 6 3.0 ± 0.60 [B] 1.4 ± 0.19 [A] 8.2 ± 0.48 [AB]6.5 ± 0.62 [B] rHPIV3-F_(B)HN_(B) 4 2.9 ± 0.28 [B] 2.0 ± 0.24 [A] 4.5 ±0.29 [D] 9.5 ± 0.65 [A] BPIV3 Ka 6 2.6 ± 0.26 [B] 1.6 ± 0.10 [A] 5.5 ±0.62 [CD] 9.2 ± 0.60 [A] ¹Monkeys were inoculated intranasally andintratracheally with 10⁵TCID₅₀ of virus in a 1 ml inoculum at each site.²The groups with 6 animals contain 4 animals each from a previous rhesusstudy (Bailly et al., 2000, supra). ³Mean of the peak virus titers foreach animal in its group irrespective of sampling day. S.E. = standarderror. ⁴Virus titrations were performed on LLC-MK2 cells at 32° C. Thelimit of detectability of virus titer was 10 TCID₅₀/ml. Mean viraltiters were compared using a Duncan Multiple Range test (α = 0.05).Within each column, mean titers with different letters are statisticallydifferent. Titers indicated with two letters are not significantlydifferent from those indicated with either letter. ⁵Nasopharyngeal swabsamples were collected on days 1 to 11 and on day 13. ⁶Trachael lavagesamples were collected on days 2, 4, 6, 8 and 10 post-infection. ⁷Thetiters on day 0 were <2.0. Day 28 was the day of challenge with wildtype HPIV3. **Two of the animals in the fHPIV3 group were infected withrHPIV3s, the virus containing two restriction enzyme recognition sitesfor the glycoprotein swap.

The rBPIV3-F_(H)HN_(H) chimeric virus replicated significantly less wellthan rHPIV3 (Table 3), and it grouped with BPIV3 in a Duncan multiplerange test. However, inspection of its pattern of replication in FIG.11B suggested that rBPIV3-F_(H)HN_(H) replicated to a level intermediatebetween that of its HPIV3 and BPIV3 parents. The interpretation thatrBPIV3-F_(H)HN_(H) replicates to a level intermediate between that ofits parents is supported by Friedman's test of consistency of ranks(Sprent, P., “A Generalization Of The Sign Test,” Applied NonparametricStatistical Method, pp. 123-126, Chapman and Hall, London, 1989,incorporated herein by reference), which indicated that the mediantiters of HPIV3, rBPIV3-F_(H)HN_(H), and BPIV3 between day 3 and day 8post infection are significantly different (d.f.2,8; p<0.05). Theobservation that the introduction of the HPIV3 F and HN proteinsresulted in an increase in the replication of BPIV3 in rhesus monkeysindicates (i) that F and HN contain one or more determinants of hostrange restriction and (ii) that one or more genetic elements of BPIV3that lie outside of the F and HN genes, e.g. the N protein, attenuatethe virus for rhesus monkeys. This confirms that the genetic basis forhost range restriction can involve multiple genes.

The Chimeric BPIV3 Bearing HPIV3 Glycoprotein Genes Induces Serum HAIAntibody to HPIV3 and a High Level of Resistance to wt HPIV3 Challenge.

rBPIV3-F_(H)HN_(H) has important features that make it a candidate liveattenuated virus vaccine against HPIV3, including attenuating genes fromBPIV3 and the antigenic specificity of HPIV3, i.e. the F and HNglycoproteins, which are the major protective antigens. Therefore, itsimmunogenicity and protective efficacy against challenge with HPIV3 weredocumented. Rhesus monkeys were immunized by infection with BPIV3 Ka,rHPIV3-F_(B)HN_(B), rBPIV3-F_(H)HN_(H), rHPIV3-N_(B), or rHPIV3. Theywere challenged 28 days later with HPIV3 JS wild type virus. Serumsamples were taken prior to the initial infection on day 0 and prior tothe challenge. BPIV3 and rHPIV3-F_(B)HN_(B) induced serum HAI antibodiesthat reacted more efficiently with BPIV3 than HPIV3, whereas theconverse was the case for HPIV3 and rBPIV3-F_(H)HN_(H). Thus, the originof the glycoprotein genes in each virus determined whether the HAIantibody response was directed predominantly against HPIV3 or againstBPIV3. The replication of challenge HPIV3 virus was significantlyreduced in the upper and lower respiratory tract of previously immunizedmonkeys (Table 4). Although the level of protective efficacy againstHPIV3 was not significantly different among the different viruses,viruses bearing HPIV3 F and HN were consistently more protective in theupper respiratory tract than were viruses bearing BPIV3 F and HN. Thisis in accordance with the higher level of HPIV3-specific serum HAIantibodies induced by viruses bearing HPIV3 F and HN.

TABLE 4 Immunization of rhesus monkeys with BPIV3/HPIV3 chimericrecombinant virus induces resistance to challenge with wild type HPIV3Mean peak virus titer³ Serum HAI antibody (log₁₀TCID₅₀/ml ± S.E.) titer(mean recip. Serum HAI antibody Immunizing Number [Duncan Grouping]⁴log₂ ± S.E.) for HPIV3 titer (mean recip. log₂ ± S.E.) for virus¹ ofanimals² Nasopharyngeal swab⁵ Tracheal lavage⁶ on the day of challengeHPIV3 28 days after challenge none 4 4.5 ± 0.33 [A] 4.5 ± 0.19 [A] <212.0 ± 0.58 [A] rHPIV3 6 2.3 ± 0.14 [B] 1.2 ± 0.20 [B] 9.5 ± 0.72 [A]11.7 ± 0.21 [A] rBPIV3-F_(H)HN_(H) 4 2.5 ± 0.25 [B] 1.0 ± 0.48 [B] 6.8 ±0.63 [BC] 10.5 ± 0.29 [AB] rHPIV3-N_(B) 6 2.3 ± 0.41 [B] 1.4 ± 0.08 [B]8.2 ± 0.48 [AB] 11.5 ± 0.22 [A] rHPIV3-F_(B)HN_(B) 4 3.0 ± 0.14 [B] 1.0± 0.0 [B] 4.5 ± 0.29 [D]  9.5 ± 0.87 [B] BPIV3 Ka 6 2.9 ± 0.26 [B] 1.3 ±0.20 [B] 5.5 ± 0.62 [CD]  9.3 ± 0.76 [B] ¹Each previously immunizedmonkey and non-immunized controls were challenged with 10⁶TCID₅₀ ofHPIV3 JS in a 1 ml inoculum at each site 28 days after immunization.²The groups with 6 animals contain 4 animals each from a previous rhesusstudy (Bailly et al., 2000, supra). ³Mean of the peak virus titers foreach animal in its group irrespective of sampling day. ⁴Virus titrationswere performed on LLC-MK2 cells. The limit of detectability of virustiter was 10 TCID₅₀/ml. Mean viral titers were compared using a DuncanMultiple Range test (α = 0.05). Within each column, mean titers withdifferent letters are statistically different. Titers indicated with twoletters are not significantly different from those indicated with eitherletter. The group of unimmunized animals were not included in the Duncananalysis at the day of challenge. ⁵Nasopharyngeal swab samples werecollected on days 3 to 8 post challenge. ⁶Trachael lavage samples werecollected on days 4, 6 and 8 post challenge. **Two animals in the rHPIV3group were infected with rHPIV3s.

Based on the foregoing examples, the invention provides for importationof BPIV genes into a virulent HPIV backbone and visa versa to yieldnovel, human-bovine chimeric PIV vaccine candidates. In exemplarychimeric recombinants disclosed in the present example,rBPIV3-F_(H)HN_(H) and its rHPIV3-F_(B)HN_(B) counterpart, replicated invitro as well as the respective parental viruses. It was also confirmedthat the F and HN exchange between the BPIV3 and HPIV3 is compatiblesince the considerably more divergent HPIV1 F and HN proteins werehighly functional in a HPIV3 background (Tao et al., J. Virol.72:2955-2961, 1998), which was evinced by the undiminished capacity ofthe chimeric viruses for replication in vitro. rBPIV3-F_(H)HN_(H)replicated in the upper respiratory tract of rhesus monkeys to a levelintermediate between that of its HPIV3 and BPIV3 parents indicating thatthe BPIV3 F and HN genes make an independent contribution to the overallattenuation of BPIV3 for primates. The overall attenuation of BPIV3virus thus is the sum of two or more genetic elements, one of which isthe set of F and HN genes and one of the others is indicated to be N.

Although BPIV3 itself is being evaluated as a vaccine virus for HPIV3(Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; and Karron etal., J. Infect. Dis. 171:1107-1114, 1995), it is only 25% relatedantigenically to HPIV3 (Coelingh et al., J. Infect. Dis. 157:655-662,1988). Thus, the immunogenicity of BPIV3 against HPIV3 will be improvedif it is modified according to the present invention to express theprotective F and HN antigens of HPIV3. rBPIV3-F_(H)HN_(H) representssuch a virus, and, in the present example, immunization of rhesusmonkeys with rBPIV3-F_(H)HN_(H) induced a higher level of antibody toHPIV3 than did immunization with BPIV3. Furthermore, rBPIV3-F_(H)HN_(H)conferred a level protection against replication of HPIV3 challenge inthe upper and lower respiratory tract that was statisticallyindistinguishable from that conferred by a previous infection withrHPIV3. Similarly, rHPIV3-N_(B), which is attenuated by the BPIV3 Nprotein but possesses HPIV3 protective antigens, also induced a highlevel of resistance to HPIV3 challenge. Despite replicating to similarlevels in rhesus monkeys, rHPIV3-N_(B) induced higher levels ofantibodies to HPIV3 than rBPIV3-F_(H)HN_(H).

rBPIV3-F_(H)HN_(H) replicates to higher levels in rhesus monkeys thanBPIV3, although it is significantly attenuated compared to HPIV3. Sincethe level of replication of BPIV3 in humans is low (Karron et al., J.Infect. Dis. 171:1107-1114, 1995), this increase is expected to be welltolerated among vaccinees. Alternatively, additional methods toattenuate human-bovine chimeric viruses of the invention are disclosedherein to ensure that the vaccine viruses replicate only to moderatelevels, for example in human infants, to prevent unacceptablerespiratory tract illness among vaccinees. Within other aspects of theinvention, the slight increase in replication of rBPIV3-F_(H)HN_(H) inprimates offers an opportunity to use rBPIV3-F_(H)HN_(H) as a vector forheterologous viral antigens such as glycoproteins of other PIVs (e.g.,HPIV1 and HPIV2), the RSV F and G glycoproteins, and the measles HAglycoprotein, which can be incorporated as added or substituted gene(s)or genome segment(s) into the attenuated HPIV3 vaccine candidate. Invarious alternative embodiments disclosed herein, the slight increase inreplication of rBPIV3-F_(H)HN_(H) in monkeys over that of BPIV3 can beoffset by the addition of foreign viral protective antigens, e.g., RSVglycoproteins, whose addition provides a selected level of attenuation.The data presented here further defined the basis for the host rangerestriction of BPIV3 for primates and identify rBPIV3-F_(H)HN_(H) as apotential vaccine candidate against HPIV3 and as a vector forheterologous viral antigens.

Microorganism Deposit Information

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under the conditions of the Budapest Treaty and designatedas follows:

Plasmid Accession No. Deposit Date p3/7(131) (ATCC 97990) Apr. 18, 1997p3/7(131)2G (ATCC 97989) Apr. 18, 1997 p218(131) (ATCC 97991) Apr. 18,1997

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and may be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

1. An isolated infectious human-bovine chimeric parainfluenza virus(PIV) comprising a major nucleocapsid (N) protein, a nucleocapsidphosphoprotein (P), a large polymerase protein (L), and a partial orcomplete PIV background genome or antigenome of a human PIV (HPIV) orbovine PIV (BPIV) combined with one or more heterologous gene(s) orgenome segment(s) of a different PIV to form a human-bovine chimeric PIVgenome or antigenome, wherein at least one of said heterologous gene(s)or genome segment(s) encodes an N protein of a BPIV3.
 2. The chimericPIV of claim 1, wherein said chimeric genome or antigenome furtherincorporates a heterologous genome segment integrated into thebackground genome or antigenome to form a chimeric gene that encodes achimeric glycoprotein.
 3. The chimeric PIV of claim 1, wherein aheterologous gene or genome segment is substituted for a counterpartgene or genome segment in a partial PIV background genome or antigenome.4. The chimeric PIV of claim 1, wherein the chimeric genome orantigenome comprises a partial or complete BPIV background genome orantigenome combined with one or more heterologous gene(s) or genomesegment(s) from a human PIV.
 5. The chimeric PIV of claim 4, wherein oneor more HPIV glycoprotein genes selected from HN and F, or one or moregenome segments encoding a cytoplasmic domain, transmembrane domain,ectodomain or immunogenic epitope thereof, is/are substituted for one ormore counterpart genes or genome segments within the BPIV backgroundgenome or antigenome.
 6. The chimeric PIV of claim 4, wherein one ormore HPIV glycoprotein genes selected from HN and F is/are substitutedto replace one or more counterpart glycoprotein genes in the BPIVbackground genome or antigenome.
 7. The chimeric PIV of claim 6, whereinboth HPIV glycoprotein genes HN and F are substituted to replacecounterpart HN and F glycoprotein genes in the BPIV background genome orantigenome.
 8. The chimeric PIV of claim 4, wherein the human-bovinechimeric PIV genome or antigenome encodes a chimeric glycoprotein havinga HPIV glycoprotein ectodomain, antigenic determinant or immunogenicepitope.
 9. The chimeric PIV of claim 8, wherein the heterologous genomesegment encodes a glycoprotein ectodomain.
 10. The chimeric PIV of claim4, wherein one or more HPIV glycoprotein genes HN and F, or a genomesegment encoding a cytoplasmic domain, transmembrane domain, ectodomainor immunogenic epitope thereof is added to or incorporated within a BPIVbackground genome or antigenome.
 11. The chimeric PIV of claim 4,wherein the chimeric genome or antigenome is further modified byaddition or substitution of one or more additional heterologous gene(s)or genome segment(s) from a human PIV within the partial or completebovine background genome or antigenome to increase genetic stability oralter attenuation, reactogenicity or growth in culture of the chimericvirus.
 12. The chimeric PIV of claim 1, wherein the bovine-humanchimeric genome or antigenome comprises a partial or complete PIV vectorgenome or antigenome combined with one or more heterologous genes orgenome segments encoding one or more antigenic determinants of one ormore heterologous pathogens.
 13. The chimeric PIV of claim 12, whereinsaid one or more antigenic determinant(s) is/are selected from HPIV1,HPIV2 or HPIV3 HN and F glycoproteins and antigenic domains, fragmentsand epitopes thereof.
 14. The chimeric PIV of claim 12, wherein one ormore gene(s) or genome segment(s) encoding one or more antigenicdeterminant(s) of HPIV2 is/are added to or substituted within thepartial or complete BPIV vector genome or antigenome.
 15. The chimericPIV of claim 12, wherein a plurality of heterologous genes or genomesegments encoding antigenic determinants of multiple HPIVs are added toor incorporated within the partial or complete BPIV vector genome orantigenome.
 16. The chimeric PIV of claim 12, wherein the vector genomeor antigenome is a partial or complete BPIV genome or antigenome and theheterologous pathogen is selected from measles virus, subgroup A andsubgroup B respiratory syncytial viruses, mumps virus, human papillomaviruses, type 1 and type 2 human immunodeficiency viruses, herpessimplex viruses, cytomegalovirus, rabies virus, Epstein Barr virus,filoviruses, bunyaviruses, flaviviruses, alphaviruses and influenzaviruses.
 17. The chimeric PIV of claim 16, wherein said one or moreheterologous antigenic determinant(s) is/are selected from measles virusHA and F proteins, subgroup A or subgroup B respiratory syncytial virusF, G, SH and M2 proteins, mumps virus HN and F proteins, human papillomavirus L1 protein, type 1 or type 2 human immunodeficiency virusgp160protein, herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG,gH, gI,gJ, gK, gL, and gM proteins, rabies virus G protein, Epstein BarrVirus gp350 protein; filovirus G protein, bunyavirus G protein,Flavivirus E and NS 1 proteins, and alphavirus E protein, and antigenicdomains, fragments and epitopes thereof.
 18. The chimeric PIV of claim16, wherein the heterologous pathogen is measles virus and theheterologous antigenic determinant(s) is/are selected from the measlesvirus HA and F proteins and antigenic domains, fragments and epitopesthereof.
 19. The chimeric PIV of claim 18, wherein a transcription unitcomprising an open reading frame (ORF) of a measles virus HA gene isadded to or incorporated within a HPIV3 vector genome or antigenome. 20.The chimeric PIV of claim 16, which incorporates a gene or genomesegment from respiratory syncytial virus (RSV).
 21. The chimeric PIV ofclaim 20, wherein the gene or genome segment encodes a RSV F and/or Gglycoprotein or immunogenic domain(s) or epitope(s) thereof.